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To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S- methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.

Plasma kallikrein has been shown to aggregate human neutrophils and release human neutrophil elastase. However, neutrophils resuspended in factor XII-deficient plasma released only 30% of the elastase compared with normal plasma. Isolated human neutrophils were aggregated in a concentration-dependent fashion by 0.06 to 0.6 U/mL factor XIIa (0.022 to 0.22 mumol/L). Factor XIIa (0.1 to 1.0 U/mL) also induced neutrophil degranulation as evidenced by a concentration-dependent release of the specific granule protein, lactoferrin, and azurophilic granule protease, elastase. The release of neutrophil elastase was biphasic, reaching 40% of maximum at 15 seconds with maximal release by 90 minutes. The active site of factor XIIa was required, since the synthetic inhibitor, D-Pro-Phe-Arg-CH2Cl, which reacts with an essential histidine, and the natural plasma inhibitor, Cl-inhibitor, which interacts with the critical serine, both inhibit by more than 90% the release of elastase. The heavy chain is also required, since factor XII fragments failed to aggregate neutrophils or stimulate degranulation. Factor XIIa (0.6 U/mL) can completely correct the defect in elastase release evident in factor XII-deficient plasma. These studies demonstrate that factor XIIa, at concentrations potentially obtainable in plasma in disease states, can activate neutrophils, and thus may participate in the inflammatory response.

The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg- p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.

Stimulation of platelets and neutrophils occurs during clinical cardiopulmonary bypass. We investigated whether the classical complement, contact, or fibrinolytic pathways are activated as potential sources of neutrophil agonists. Using enzyme-linked immunosorbent “sandwich” assays specific for C1s-C1-and kallikrein-C1- inhibitor complexes respectively, we found that there was a modest increase in plasma levels of each complex after clinical cardiopulmonary bypass was completed. The increased concentration of enzyme-inhibitor complexes reverted to baseline within 24 hours. Since these complexes are cleared in vivo, we measured their formation by assaying their plasma levels during in vitro simulated extracorporeal circulation. Over a period of two hours, C1s-C1-inhibitor complexes rose from a baseline of 2 +/- 1 nmol/L to 21 +/- 2 nmol/L, and kallikrein-C1-inhibitor complexes rose from 2 +/- 1 nmol/L to 25 +/- 5 nmol/L. However, there was no evidence of either in vivo or in vitro plasmin-alpha 2-plasmin-inhibitor complex formation. These results indicate that the pathways of classical complement and contact activation, but probably not fibrinolysis, may be associated with neutrophil activation seen during clinical cardiopulmonary bypass.

We have isolated and probed the mechanism of action of two naturally occurring antibodies (Baltimore and Winston-Salem) against factor XI (FXI), that developed in patients congenitally deficient in FXI after replacement therapy. Purification on immobilized protein A and neutralization with monospecific antibodies against IgG heavy and light chain subtypes indicated that both antibodies were of restricted heterogeneity. Both Winston-Salem (IgG3 kappa) and Baltimore (IgG1 kappa) completely inhibited FXI coagulant activity at titers of 200 and 8 Bethesda units, respectively. Immunoaffinity columns prepared from each antibody were able to bind the heavy but not the light chain of reduced and alkylated activated FXI (FXIa). The activation of purified FXI by activated bovine factor XII (FXIIa), a reaction independent of high molecular weight kininogen (HK), was not inhibited by either antibody. The active site on the FXIa light chain was unaffected by either patient's IgG, as measured by its amidolytic activity. In contrast, one antibody (Baltimore) or its Fab' blocked the surface- mediated proteolytic activation of FXI by human FXIIa in a concentration-dependent fashion by preventing its binding to HK, but had no effect on the rate of activation of FIX by FXIa. In contrast, the other antibody (Winston-Salem) or its Fab' inhibited the activation of FIX by FXIa in a concentration-dependent fashion but did not inhibit binding of FXI to HK. We conclude that each of these two naturally occurring antibodies is directed against a specific, separate, and distinct epitope located in the heavy chain of FXIa, one near or at the domain essential for the activation of FIX by FXIa and the other close to the domain required for binding to HK.

C1 inhibitor (C1-INH) and alpha 2-macroglobulin (alpha 2M) account for over 90% of the inactivation of purified plasma kallikrein by normal human plasma. The rate of kallikrein inactivation is also dependent on the presence of high molecular weight kininogen (HMWK), which forms a reversible complex with kallikrein protecting the active site of the enzyme against inhibitors. By selectively inactivating alpha 2M with methylamine, and eliminating the protective effect of HMWK by dilution, the inactivation of kallikrein by plasma became almost exclusively dependent on C1-INH. Functional C1 inhibitor was assessed by measuring the pseudo-first-order rate constant for the inactivation of kallikrein by diluted methylamine-treated plasma in 29 individuals, including 11 controls, 11 oral contraceptive users, 5 patients with classical hereditary angioedema (HAE), and 2 patients with variant HAE. Over a wide range of concentrations, an excellent correlation (r = 0.90) was observed between functional and antigenic C1-INH among controls, oral contraceptive users, and patients with classical HAE. This new functional assay for C1-INH can be performed in less than 3 hr with commercially available reagents. Therefore, this assay will be helpful for the diagnosis and management of conditions associated with the deficiency of C1-INH, such as HAE.

Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.

After being envenomated by the timber rattlesnake, a patient was found to have a platelet count of 5000 per microliter, prothrombin time and activated partial thromboplastin time both greater than 150 sec, plasma fibrinogen 0 mg/dl, and fibrinogen split products 2560 microgram/ml. However, this patient did not appear to have acute disseminated intravascular coagulation since coagulation factors II-XII were normal. We postulated that this venom contained, in addition to a fibrinogen clotting enzyme, a platelet activating protein, Crotalocytin. Crotalocytin was purified from crude timber rattlesnake venom by Sephadex G-100 gel-filtration, low ionic strength precipitation, and DEAE-A50 Sephadex chromatography. By sodium dodecyl sulfate gel electrophoresis and gel-filtration Crotalocytin was a single chain polypeptide, molecular weight 55,000. Thrombocytopenia after timber rattlesnake bite appeared to be due to a protein that directly activated platelets. Timber rattlesnake bite mimicked the clinical presentation of disseminated intravascular coagulation.

Human and rhesus monkey platelets secrete at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4). Neither of these proteins showed species-related antigenic differences. As determined by radioimmunoassay, the levels of PF4 and LA-PF4 antigen per 10(9) monkey platelets amounted to 10.7 and 20.3 microgram, respectively. One milliliter of monkey plasma prepared from blood collected into an anticoagulant composed of EDTA, prostaglandin E1, and theophylline solution contained 22.4 ng LA-PF4 and 8.0 ng PF4. Concentrations of these two platelet-specific proteins in monkeys closely resembled levels found in human platelets and plasma. Infusion of prostacyclin (PGI2) (100 or 300 ng/kg/min) into monkeys for 15 min resulted in a significant decrease of plasma levels of LA-PF4 antigen and of PF4 by 40%--60% (p < 0.0001). This decrease was related to the inhibitory effect of PGI2 on the secretion of platelets stimulated by a catheter or by venipuncture. Longer infusion of PGI2 did not produce further significant change. The supernate obtained after aggregation of human platelets stimulated by thrombin was injected into monkeys receiving PGI2 infusion. The disappearance of LA-PF4 antigen in monkey plasma followed a biphasic exponential curve with half-lives for the fast and slow components of 8.4 and 63 min. PF4 disappeared faster but followed the same pattern (half-lives for the fast and slow component of 2.1 and 70 min). Analysis of the experimental data suggests that the low levels of secreted platelet proteins in monkey plasma are related to their minimal in vivo release and to their rapid clearance.

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.

The objective of this study was to determine the role of the kallikrein- kinin system in healthy humans after intravenous administration of either Escherichia coli endotoxin or saline. We studied a total of 18 healthy nonsmoking volunteers, 23 to 38 years old, in an open-label study at the Critical Care Medicine Department, Clinical Center, National Institutes of Health (Bethesda, MD) in which some of the patients served as their own controls. After baseline data collection, the subjects received intravenously either E coli endotoxin (n = 15, 4 ng/kg of body weight) or saline (n = 8, controls). Signs, symptoms, systemic blood pressure, factor XII, plasma prekallikrein (PK), factor XI (FXI), antithrombin III (AT-III), high molecular weight kininogen (HK), and alpha 2-macroglobulin-kallikrein complexes were measured at baseline and 1, 2, 3, 5, and 24 hours after injection of either saline or endotoxin. After infusion of endotoxin, we found the functional plasma levels of FXI decreased at 2 hours (P < .05) and at 5 hours (P < .05). Functional PK was significantly depressed by 2 hours (P < .05), at 5 hours (P < .05), and at 24 hours (P < .01), whereas the PK antigen was only low at 5 hours (P < .05). These changes were accompanied by a significant increase in circulating alpha 2-macroglobulin-kallikrein complexes at 3 hours (P < .05) and 5 hours (P < .01). No significant changes occurred in the plasma levels of factor XII or HK. We concluded that clinical response to intravenous endotoxin in healthy human volunteers is associated with activation of the kallikrein-kinin systems. Further investigation is needed with specific inhibitors of the kallikrein-kinin system to define its primary or secondary role in the endotoxin-mediated reactions.

Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1–536) as well as four other cDNAs (nt 1459–1632, nt 1765–1986, nt 2152–2538, and nt 2978–3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.

Coagulation factor V (FV) has been shown to be synthesized in both the liver and megakaryocytes. We now present evidence that FV can be covalently crosslinked by an enzyme originating from megakaryocytes to form polymeric multimers of factor V. The guinea pig megakaryocyte enzyme appears to be factor XIIIa since the FV-crosslinking activity (1) had an absolute requirement for Ca++, (2) was completely inhibited by iodoacetamide, 5,5'-dithiobis- (2-nitrobenzoic acid), p- chloromercuribenzene sulfonic acid, and N-ethylmaleimide, all known alkylators of the thiol group at the active site of the factor XIIIa, (3) was blocked by known pseudoamine donor substrates of factor XIIIa including dansylcadaverine and putrescine, and (4) could be directly demonstrated in the guinea pig megakaryocyte lysate by a specific activity staining procedure. No tranglutaminase was detected in guinea pig megakaryocytes in contrast to red cells and liver. A similar pattern of covalent crosslinking of human FV by purified activated human plasma factor XIII was also demonstrated. Analysis of the crosslinked products of FV formed by the guinea pig enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates the formation of intermediate as well as higher molecular weight polymers, suggesting that the crosslinking is a stepwise polymerization process.

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.

Platelets are known to process human factor V during secretion and/or membrane binding. We studied the functional and structural changes produced in human factor V by purified human platelet calpain (calcium- activated thiol protease) and compared the alterations with those induced by thrombin. A maximum increase in coagulant activity of 2.5- fold was observed when factor V (1 U/mL, 33 nmol/L) was incubated with calpain (0.03 U/mL, 2.7 nmol/L) in comparison with a 8.8-fold increment for alpha-thrombin (0.7 U/mL, 8 nmol/L) at 25 degrees C. Thrombin additions to reactions initiated by calpain resulted in further activation comparable to that of thrombin alone, whereas the subsequent addition of calpain had no effect on the extent or pattern of the activation of factor V by thrombin. The cleavage pattern of factor V produced by these two enzymes are distinctly different. Although thrombin activation eventually results in four final components designated C1 (150 kd), D (105 kd), E (71 kd), and F1F2 (71 to 74 kd), calpain yields initial components of 200 kd and 160 kd within one minute. Further digestion of the 200 kd species by calpain gives rise first to a polypeptide of 160 kd that is converted to a 140 kd and a 120 kd species by two minutes with an increase in coagulant activity. Immunoblotting of these fragments with the monoclonal antibody (MoAb) B10 directed to factor V and the thrombin-generated C1 fragment yields results demonstrating a common epitope in these calpain-generated components of 200, 160, 140 and 120 kd. The degradation of the initial 160 kd polypeptide gives rise to polypeptides of 100 and 65 kd, both undetectable on immunoblotting with MoAb B10. The 130, 87, 58, and 48 kd components are of less certain origin. Thus, platelet calpain generates a complex but reproducible cleavage pattern different from thrombin that may explain the partial activation observed. Nevertheless, calpain processing may play a role in early hemostatic reactions involving platelets before the appearance of the first thrombin molecule.

The relative roles of platelet autacoids such as adenosine diphosphate (ADP), prostaglandin endoperoxides, and thromboxane A2 (TXA2) in collagen-induced platelet activation are not fully understood. We reexamined this relationship using the ADP affinity analogue, 5'-p- fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies a receptor for ADP on the platelet surface, thereby inhibiting ADP- induced platelet activation. Collagen-induced shape change, aggregation, and fibrinogen binding were each fully inhibited under conditions in which FSBA is covalently incorporated and could not be overcome by raising the collagen used to supramaximal concentrations. In contrast, TXA2 synthesis stimulated by collagen under conditions that produced maximum aggregation was only minimally inhibited by FSBA. Since covalent incorporation of FSBA has been previously shown to specifically inhibit ADP-induced activation of platelets, the present study supports the contention that ADP is required for collagen-induced platelet activation. Under similar conditions, indomethacin, an inhibitor of cyclooxygenase, inhibited collagen-induced shape change, indicating that endoperoxides and/or TXA2 also play a role in this response. Shape change induced by low concentrations (10 nmol/L) of the stable prostaglandin endoperoxide, azo-PGH2, was also inhibited by FSBA. These observations indicate a role for ADP in responses elicited by low concentrations of endoperoxides. However, at higher concentrations of azo-PGH2 (100 nmol/L), inhibition by FSBA could be overcome. Thus, the effect of collagen apparently has an absolute requirement for ADP for aggregation and fibrinogen binding and for both ADP and prostaglandins for shape change. Aggregation and fibrinogen binding induced by prostaglandin endoperoxides also required ADP as a mediator, but ADP is not absolutely required at high endoperoxide concentration to induce shape change.

High molecular weight (HMW) kininogen, the cofactor for activation of the contact system of plasma proteolysis, transports and optimally positions prekallikrein and factor XI on a negatively charged surface, allowing those zymogens to be activated by surface-bound factor XIIa. HMW kininogen circulates in plasma as a procofactor that, after cleavage by kallikrein or factor XIIa, gains ability to bind to the surface. The mechanism responsible for this increased affinity for the surface is unknown. We hypothesized that modification of arginine residues may prevent cleavage of HMW kininogen, since the initial kallikrein-induced cleavage sites on the HMW kininogen molecule are at the NH2 terminal and the COOH terminal of the bradykinin-containing portion of the molecule, each of which contains arginine. We found that modification with butanedione of four arginine residues in the HMW kininogen molecule prevented bradykinin release, which results from cleavage of HMW kininogen. Furthermore, HMW kininogen coagulant activity was lost, in proportion to the degree of arginine modification, until 6.6 residues had been modified. Complex formation with prekallikrein, however, was found to be uneffected by the modification of modified HMW kininogen. To account for the loss of coagulant activity, we also examined the ability of modified HMWKa (active cofactor) to bind to an activating surface. The affinity of modified HMWKa for kaolin was tenfold less than the affinity of unmodified HMWKa. These data suggest that arginine residues play a critical role in the ability of HMW kininogen to function as an activation cofactor, both by preventing the cleavages that produce HMWKa as well as by decreasing the affinity of HMWKa for the surface.

Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)- 1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(- 8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine- directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15- aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

Human plasma kallikrein consists of an N-terminal heavy chain of molecular weight (mol wt) 52,000, linked by disulfide bonds to two light chain variants (mol wt 36,000 or 33,000). Although the active catalytic site of kallikrein resides on the C-terminal light chain, the role of the N-terminal heavy chain is less clear. We therefore studied an enzyme designated beta-kallikrein, containing a single cleavage in the heavy chain (mol wt 28,000 + 18,000) and compared it to the enzyme, alpha-kallikrein, with an intact heavy chain. The rates of inactivation by C1 inhibitor of plasma alpha- and beta-kallikreins were kinetically identical, as measured by residual amidolytic activity, after various times of incubation with the inhibitor. Both enzymes reacted completely with C1 inhibitor after 18 hours and formed identical C1 inhibitor- kallikrein complexes of mol wt 195,000. The rate of activation of factor XII by alpha-kallikrein and beta-kallikrein was similar. In contrast, the rate of cleavage of high molecular weight kininogen (HMWK) by alpha-kallikrein was at least fivefold faster and the ratio of coagulant activity to amidolytic activity was fourfold greater than for beta-kallikrein. Plasma alpha-kallikrein, at concentrations potentially achievable in plasma, induced aggregation of neutrophils, but beta-kallikrein failed to elicit this response. In addition, human neutrophils pretreated with cytochalasin B released 2.46 +/- 0.10 microgram/10(7) cells of elastase antigen, but beta-kallikrein released only 0.25 +/- 0.10 micrograms/10(7) cells. These observations suggest that cleavage of the heavy chain influences the rate of cleavage of HMWK and decreases its coagulant activity. Moreover, an intact heavy chain appears to be requisite to support the ability of kallikrein to aggregate neutrophils and release elastase.

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.

An 82-year-old woman presented with extensive hematomas and melena associated with markedly decreased plasma factor V coagulant activity (FV:C). Using a competitive enzyme-linked immunosorbent assay developed in our laboratory, we made serial measurements of factor V antigen (FV:Ag) in plasma and found it to be normal or elevated. The patient's plasma was demonstrated to contain an IgG antibody that could neutralize FV:C in normal plasma. The antibody was of restricted heterogeneity (IgG1, IgG2,kappa). Circulating immune complexes containing antibody to factor V and FV:Ag were demonstrated directly in the plasma by immunoelectrophoresis with polyclonal monospecific antibody and with a monoclonal antibody using an enzyme-linked immunosorbent assay. Presence of neutralizing antibody could be demonstrated in vitro even at times when FV:C was within normal limits by heat inactivation of FV:C. Treatment with plasma and platelet transfusions as well as plasmapheresis induced definite but transient elevation of FV:C. Steroid therapy lowered the neutralizing antibody concentration and produced a rapid and persistent elevation of FV:C during two separate hospitalizations. This report describes a patient in whom levels of FV:Ag have been serially measured, and the presence of circulating immune complexes consisting of factor V and a neutralizing antibody have been directly demonstrated.

An immunocytochemical study was performed to examine the cellular localization and the subcellular distribution of kininogens in human blood cells. Kininogens were visualised using the immunogold-silver staining method and confocal scanning laser microscopy. We confirmed the existence of high molecular weight kininogen in human neutrophils and describe for the first time the presence of low molecular weight kininogen on these cells. Both high and low molecular weight kininogens were restricted to the neutrophils where they localized as clusters of immunogold particles on the cell membrane. No labeling was observed intracellularly in organelles such as mitochondria, endoplasmic reticulum, and azurophilic or specific granules after permeabilization of the neutrophils with Triton X-100, a procedure that permitted the visualization of elastase in the azurophilic granules. Clusters of high molecular weight kininogen molecules attached to the neutrophil surface could serve as receptors for plasma kallikrein and/or be the source of substrate for a discrete and circumscribed formation of kinins that may in turn facilitate the local diapedesis of neutrophils and the transudation of plasma constituents during acute inflammation.

Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the alpha 2- macroglobulin-kallikrein (alpha 2M-Kal) complex using an F(ab')2 derivative of a monospecific polyclonal antibody against alpha 2 M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4 degrees C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for alpha 2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of alpha 2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for alpha 2M- Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks.

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)- induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin- induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma- thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma- thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

Controversy exists in the literature concerning the potentiating effect of heparin on the inactivation rate of factor XIa by antithrombin III (AT III) in both purified systems and in plasma. We have analyzed the factors that could influence this reaction and found that ionic strength of the medium, as well as the type and concentration of the heparin preparations accounted for the major discrepancies in the literature. At I = 0.43 N, a preparation of bovine lung heparin at 1 U/mL did not augment the inactivation rate of factor XIa by inhibitors in plasma or by purified AT III. However, when ionic strength was decreased, a progressive increase in the potentiating effect was observed, reaching 6.5-fold at I = 0.15 N. At saturating concentrations of heparin, which results in the formation of 100% AT III-heparin complex, (greater than ten-fold molar excess over AT III) in purified systems, all heparin preparations (porcine, bovine, low molecular weight [LMW], and high affinity) yielded an approximately 30-fold augmentation of the factor XIa inactivation rate. However, when heparin was less than saturating, we observed that various heparin preparations affected the AT III-induced inactivation of factor XIa to different degrees even though they exhibited the same inhibitory activity (1 U/mL) against thrombin. This variation resulted from differences in the number of AT III binding sites in each heparin preparation, despite a similar Kd for each. Addition of high molecular weight kininogen (HK) to AT III-heparin complexes did not enhance their ability to inhibit factor XIa, and high concentrations of HK decreased the inactivation rate. A high therapeutic dose of heparin only permits the formation of 2.5% to 16.5% of the AT III-heparin complexes that can be achieved at saturation. We observed that 1 U/mL heparin (bovine lung heparin) (high therapeutic concentration) in virtually undiluted plasma only accelerated the inactivation rate of factor XIa (in the absence of other active enzymes) less than two-fold. These new observations further support our previous conclusion that therapeutic levels of heparin have little to no influence on the inactivation rate of factor XIa in plasma.

Of five IgGI/k murine monoclonal anti-human prekallikrein antibodies produced (MAbs), MAb 13G11 was selected for studying interaction of prekallikrein with factor XII and high-mol-wt kininogen (HMWK) during activation on a surface. Immunoblots from sodium dodecyl sulfate (SDS) gels showed that this MAb recognizes two variants (88 kd and 85 kd) of prekallikrein and kallikrein both in purified proteins and normal plasma. Under reducing conditions, kallikrein exhibits the epitope on the heavy chain but not on the light chains. Preincubation of MAb 13G11 with prekallikrein (added to prekallikrein-deficient plasma) or with normal plasma inhibited surface activation of prekallikrein 60% to 80%, as judged by amidolytic and coagulant assays. In normal plasma, inhibition by the Fab fragments was 87% of that with the entire MAb. Inhibition was not by competition between the MAb and HMWK, since neither binding of 13G11 to prekallikrein (coated on microtiter plates) was inhibited by an excess of HMWK, nor was hydrolysis of HMWK by kallikrein inhibited by 13G11. Using purified proteins in a system mimicking contact activation, inhibition by 13G11 of prekallikrein activation by factor XIIa, HMWK, and kaolin present was approximately 80%. Decreased inhibition (55% to 25%) occurred without HMWK or when kallikrein was used instead of prekallikrein. Kallikrein activity was not inhibited by 13G11 Fab fragments. These results indicate that the effect of 13G11 in plasma was neither dissociation of prekallikrein- HMWK complex nor a direct effect on kallikrein activity. Similar to the results in plasma, activation of prekallikrein, HMWK present, by factor XIIa bound to kaolin, was inhibited approximately 70% by 13G11. The results suggest a previously unrecognized site on the prekallikrein (heavy chain) required for its interaction with factor XIIa, either shared with the 13G11 epitope or located in very close proximity. The inhibition of kallikrein by intact 13G11 indicates that its binding site on the heavy chain is sterically related to the active site (light chain).

The transient detection of fibrinogen on surfaces has been described (Vroman effect) and high-mol-wt kininogen (HK) has been shown to play a role in this reaction. In this study, we attempted to identify the form of HK responsible for preventing detection of the fibrinogen initially adsorbed from plasma to various artificial surfaces and to determine if other plasma components were involved. We compared 125I-fibrinogen adsorption in the presence of normal plasma to plasma deficient in specific proteins. On all surfaces tested, we found that fibrinogen was displaced from the surface. The extent of displacement was greatly reduced, however, but not eliminated in HK-deficient plasma. Factor XII- deficient plasma also showed reduced fibrinogen displacement. These data indicate that HK can actually displace fibrinogen; however, factor XII, or a factor XII-mediated reaction also appears to be necessary for this displacement to occur. Furthermore, when normal plasma was first subjected to extensive contact activation by dextran sulfate, during which the HK was extensively degraded to components smaller than the light chain (as assessed by Western blotting), we observed greatly reduced displacement of fibrinogen. Extensive contact activation of Factor XI-deficient plasma failed to show low-mol-wt derivatives, however, and displacement of fibrinogen was similar to normal plasma that had not undergone extensive activation. These data indicate that HKa (active cofactor produced during contact activation by factor XIIa or kallikrein) is primarily responsible for displacing fibrinogen, and that HKi (inactive cofactor generated by factor XIa) cannot displace fibrinogen. The fibrinogen from all plasma samples looked similar by Western blot analysis, suggesting that fibrinogenolysis was not a component of the Vroman effect. In addition, experiments performed with plasma prechromatographed on lysine agarose showed that a lysine- agarose adsorbable protein may be minimally involved in fibrinogen desorption and a synergism may exist between HK and that protein.

Previous reports have indicated that the nucleotide affinity analog 5'- p-fluorosulfonylbenzoyl adenosine (FSBA) at concentrations between 40 and 100 mumol/L and at times greater than 20 minutes covalently modifies a single protein component on the external platelet membrane surface and that adenosine diphosphate (ADP) protects against this reaction. That this protein is an ADP receptor linked to platelet activation is shown by FSBA inhibition of ADP-mediated platelet shape change, aggregation, and fibrinogen receptor exposure. In this report, further evidence for the interaction of FSBA with the ADP receptor on platelets is provided by the observation that FSBA at high concentrations (100 to 500 mumol/L) behaves as a weak agonist to produce platelet shape change within one minute as detected by spectroscopic assay and scanning electron microscopy with concomitant phosphorylation of the light chain of platelet myosin. The specificity of FSBA as an agonist is demonstrated by inhibition of FSBA-induced shape change by ATP and the covalent incorporation of SBA as well as the failure of 5'-fluorosulfonylbenozoyl guanosine (FSBG) to cause shape change. In contrast, incubation of platelets with low concentrations of [3H]-FSBA (40 mol/L) is not associated with stimulation of platelet shape change or myosin light chain phosphorylation.

We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

Factor XIa catalyzes an important reaction in the early phase of blood coagulation by converting factor IX to an active enzyme (factor IXa). Although antithrombin-III, an inhibitor of factor XIa, normally accounts for only one-sixth of the plasma inhibitory activity against factor XIa, its effectiveness has been reported to be enhanced by heparin. We have reinvestigated the ability of heparin to potentiate factor XIa inhibition by both purified antithrombin-III and plasma using synthetic tripeptide amide substrates as well as a coagulant assay. No increase in the inactivation rate of factor XIa amidolytic activity by purified antithrombin-III was observed in the presence of therapeutic heparin concentrations (1 U/ml), although inhibition of the amidolytic activity of thrombin by purified antithrombin-III was enhanced at least 20-fold by the same concentration of heparin. Furthermore, despite the ability of heparin (1 U/ml) to increase the inactivation rate of thrombin by plasma, no acceleration of the rate of inhibition of factor XIa by plasma was observed. Similar results were found when the inhibition of factor XIa was monitored with a coagulant assay after first removing the heparin. Only at heparin concentrations of 5 and 10 U/ml, was a 2- and 4-fold increase in the inactivation rate of factor XIa by purified antithrombin III observed. Therefore, in both purified systems as well as plasma, heparin, at concentrations observed in clinical practice, does not accelerate the inactivation rate of human factor XIa by antithrombin-III.

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.

Using the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCl, an amidolytic assay was designed to measure prekallikrein in plasma. At a substrate concentration of 1 mM (Km = 0.2 mM), the amidolysis of purified kallikrein at 1 coagulant unit/ml was observed to be 2.47 mumole/min/ml. Conditions for plasma prekallikrein activation were optimized to approach complete activation when compared to the amidolytic activity of the purified plasma kallikrein. Plasma treated with chloroform to destroy inhibitors of kallikrein was activated with dilute kaolin (final concentration 1 mg/ml) for 1 min at 25 degrees C. Activated plasma prekallikrein had 78% (1.92 mumole/min/ml) of activity of purified kallikrein at plasma concentration. Comparison of this amidolytic assay with immunochemical, esterolytic, and coagulant assays of three subject populations (normals, women on birth control pills, and patients with hepatocellular disease) showed good correlation both in normals and in the patient groups between the amidolytic and esterolytic assays (r = 0.89). Each enzymatic assay correlated with the immunochemical assay (r = 0.72, r = 0.68, respectively). However, comparison of each of these assays with the coagulant assay showed no significant correlation due to the large inherent error of the latter assay. This standardized plasma prekallikrein amidolytic assay should facilitate studies of plasma prekallikrein concentration in physiologic and pathologic conditions and help identify activation of the contact phase of coagulation in disease states.

We studied the mechanism by which complement activated by anti-P1A1 antibody elicits the platelet release reaction. Anti-P1A1 antibody mediates its action through the classic complement pathway, and its effect depends on the concentration of IgG antibody on the platelet surface. At relatively high concentrations of anti-P1A1 antibody the release reaction was mediated by a mechanism in part independent of extracellular ADP and metabolic energy and inhibited by only high concentrations of PGE1. However, at lower concentrations of anti-P1A1 antibody the release reaction was dependent on metabolic energy and ADP, and the concentration of PGE1 required to inhibit platelet release was similar to that required to inhibit ADP-induced release. The cyclooxygenase inhibitor acetylsalicylic acid inhibited the release reaction at all nonlytic antibody levels studied. None of the agents studied inhibited the induction of platelet lysis by very high concentrations of anti-P1A1 antibody, and no effect of antibody on platelet 14C-serotonin uptake was observed at antibody concentrations that did not mediate direct in vitro alteration. These studies suggest the possible use of pharmacologic agents in modifying some complement- mediated platelet alterations.

The effect of heterologous anti-human platelet antibody on human platelet function was examined in the presence and absence of whole plasma as an in vitro model for antibody-induced immune damage to cells. Heterologous IgG anti-human platelet antibody mediated platelet aggregation and released serotonin from both platelets in plasma and from platelets isolated by gel filtration and increased the availability of platelet acid phosphatase in a dose-response fashion. Anti-platelet antibody failed to release beta-glucuronidase (lysosomal enzyme marker) or cause lactic dehydrogenase loss (cytolysis). The effect of the antiplatelet antibody on platelets proceeded in the absence of complement. The active molecule in the anti-platelet antiserum was isolated in the IgG fraction and all three indi cators of platelet injury were mediated by purified monomeric IgG. Thrombin was not required for the antibody-mediated effects, as three thrombin inhibitors failed to block the reaction. EDTA was an effective inhibitor, suggesting a cation requirement; however, as little as 38 muM calcium was sufficient for effective platelet aggregation and release. The inability of acetylsalicylic acid to inhibit the effect of the antiplatelet antibody suggests that heterologous antibody (IgG) induced platelet alteration proceeds by a different mechanism than that mediated by ADP and epinephrine and does not involve endogenous platelet prostaglandin synthesis.

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.

In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates.

Although coagulation factor Xa requires Ca2+ for binding to phospholipid, factor V, the other protein component in the prothrombinase enzyme complex, binds tightly to phospholipid in the absence of Ca2+. To explore the possibility that calcium might be present in the fact V molecule, the effect of several chelators, including oxalate, citrate, pyrophosphate, and EDTA, on factor V activity has been studied. A time- and concentration-dependent inhibition of factor V which reflects the respective association constants for calcium of each chelator is observed. The inhibition can be prevented by the prior addition of calcium and manganese but not magnesium. Reversal of the activity loss can be accomplished at high protein concentrations by the addition of calcium, the removal of the chelator by gel filtration, or an increase in temperature. Factor V contains 1 g atom of calcium per 300,000 daltons which is not removed by incubation with EDTA under nondenaturing conditions. Thus, the inhibition by EDTA is due to binding to calcium associated with factor V. In 8 M urea, EDTA can remove over 80% of the calcium, demonstrating the importance of the native structure in maintaining the calcium binding site. Prior binding of phospholipids to factor V prevents inhibition by EDTA. The results suggest that phospholipids complex at the calcium site on the factor V metallopretein.