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Two likely mechanisms for the initiation of arterial platelet thrombus formation under conditions of elevated fluid shear stresses are: (1) excessive adhesion and aggregation of platelets from rapidly flowing blood onto the exposed sub-endothelium of injured, atherosclerotic arteries; or (2) direct, fluid shear stress-induced aggregation of platelets in constricted arteries with intact endothelial cells. Mechanism (1) was simulated using a parallel plate flow chamber, fibrillar collagen type I-coated slides, and mepacrine-labeled (fluorescent) platelets in whole blood anticoagulated with citrate, hirudin, unfractionated porcine heparin, or low molecular weight heparin flowing for 1 to 2 minutes at wall shear rates of 100 to 3,000 seconds-1 (4 to 120 dynes/cm2). The precise sequence of interactions among von Willebrand factor (vWF), glycoprotein (GP)Ib, and GPIIb-IIIa during platelet adhesion and subsequent aggregation were resolved by direct real-time observation using a computerized epifluorescence video microscopy system. Adhesion at high shear rates was the result of the adsorption of large vWF multimers onto collagen and the binding of platelet GPIb to the insolubilized vWF. Aggregation occurred subsequently and required the binding of ligands, including vWF via its RGD binding domain, to GPIIb-IIIa. Mechanism (2) was modeled by producing shear stresses of 90 to 180 dynes/cm2 in a rotational cone and plate viscometer, which aggregates platelets from platelet-rich- plasma (PRP) anti-coagulated with citrate, hirudin, or either type of heparin in reactions that require large vWF multimers, Ca2+, adenosine diphosphate, and both GPIb and GPIIb-IIIa. Both vWF-mediated shear- aggregation in PRP and platelet-collagen adhesion in flowing whole blood (anticoagulated with citrate and hirudin) are inhibited by two potentially useful anti-arterial thrombotic agents: polymeric aurin tricarboxylic acid (ATA; 28.5 to 114 micrograms/mL), which binds to vWF and inhibits its attachment of GPIb, and a recombinant vWF fragment (rvWF445–733; 30 to 200 micrograms/mL) that binds to platelet GPIb (in the absence of any modulator) and blocks attachment of vWF multimers. Unfractionated heparin, but not low molecular weight heparin, apparently binds to rvWF445–733 and counteracts the inhibitory effects of the vWF fragment in vitro on shear-aggregation and platelet-collagen adhesion.

Thrombotic thrombocytopenic purpura (TTP) is a disorder of systemic platelet aggregation. Evidence has accumulated that the aggregating agonist in TTP of all types is likely to be von Willebrand factor, especially unusually large von Willebrand factor multimers derived from endothelial cells. Recent evidence indicates that a metalloproteinase involved in von Willebrand factor breakdown is not present in adequate amounts in children with chronic relapsing TTP. Chronic relapsing TTP is, therefore, likely to be a congenital deficiency of von Willebrand factor metalloproteinase. In adults with single episode or intermittent types of TTP, the von Willebrand factor metalloproteinase is inhibited by autoantibodies that are present either transiently or intermittently in patient blood. Single episode and intermittent types of TTP in adults are, therefore, autoimmune processes of a short-term and recurrent nature, respectively.

Thrombotic thrombocytopenic purpura (TTP) is a disorder of systemic platelet aggregation. Evidence has accumulated that the aggregating agonist in TTP of all types is likely to be von Willebrand factor (vWf), especially unusually large vWf multimers derived from endothelial cells. Recent evidence indicates that a metalloproteinase involved in vWf breakdown is produced in inadequate amounts in children with chronic relapsing TTP. Chronic relapsing TTP is, therefore, likely to be a congenital enzyme deficiency. In adults with single episode or intermittent types of TTP, the vWf metalloproteinase is inhibited by autoantibodies that are present either transiently or intermittently in patient blood. Single episode and intermittent types of TTP in adults are likely to be short-term or recurrent autoimmune processes, respectively.

Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/microl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet alpha-granular protein, P-selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from alpha-granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf-platelet interactions are involved in the platelet clumping process that characterizes TTP.

The vascular subendothelium contains macromolecular structures called microfibrils. Type VI collagen is one protein found in microfibrils that supports platelet adhesion and aggregation and we have previously evaluated the roles of platelet receptors and vWf involved in these processes under physiological shear conditions. Here we investigate the ability of fibrillin containing elastic microfibrils to support mural thrombus formation. Our results show that elastic microfibril surfaces support platelet adhesion under low shear conditions at a level similar to collagen VI tetramers. However, the degree of aggregation on the elastic microfibril surface is much higher. Both adhesion and aggregation were shown to be mediated by the GPIIb-IIIa platelet receptor. Elastic microfibrils do not support the formation of mural thrombi under high shear conditions. These results suggest roles for both collagen VI and fibrillin containing elastic microfibrils in modulating the platelet response to blood vessel injury.

Recent studies indicate that CRTTP patients have excessive shear stress-induced platelet aggregation that is associated with the presence of ULvWF multimers in their plasma and increased vWF-binding to their platelets by flow cytometry. In these CRTTP patients, relapses, excessive shear-aggregation and the presence in their plasma of ULvWF forms are all reversed by the infusion of normal FFP or substances devoid of the largest vWF multimers found in plasma (cryoprecipitate-poor plasma or cryosupernatant; solvent/detergent-treated plasma) without the need for concurrent plasmapheresis. This constellation of observations, along with reports of increased vWF in TTP platelet thrombi, increases the probability that ULvWF multimers derived from injured or abnormal endothelial cells induce aggregation during TTP episodes in high-shear regions of the microcirculation.

Type VI collagen is a subendothelial constituent that binds von Willebrand factor (vWF) and platelets. The interaction of platelets with type VI collagen and the roles of platelet glycoprotein (GP) receptors and vWF were studied under flow conditions using epi-fluorescent videomicroscopy coupled with digital image processing. We found that surface coverage was less than 6% on collagen VI at a relatively high-wall shear rate (1,000 s-1) and was approximately 60% at a low-wall shear rate (100 s-1). The molecular mechanisms involved in low-shear platelet binding were studied using monoclonal antibodies to platelet GPIb and GPIIb-IIIa, and polymeric aurin tricarboxylic acid. Anti-GPIIb-IIIa was the most effective in eliminating adhesion (surface coverage, 0.8%), followed by anti-GPIb (4.3%), and ATA (12.6%). Experiments with von Willebrand disease blood indicate that vWF is involved in platelet adhesion to collagen VI at 100 s-1. In the absence of vWF, there may be direct binding of platelet GPIIb-IIIa complexes to collagen VI. Adhesion and aggregation on collagen VI are different in shear rate dependence from collagen I. Our results suggest a possible role for collagen VI and vWF in platelet adhesion and aggregation in vascular regions with low shear rates.

The clinical manifestations of severe preeclampsia and thrombotic thrombocytopenic purpura are similar. Patients with thrombotic thrombocytopenic purpura have been demonstrated to have larger than usual von Willebrand factor multimers. Serial serum samples were taken from patients with severe preeclampsia, all with platelet counts less than 50,000. Von Willebrand factor multimeric patterns were normal in all the patients except one, who later proved to have chronic relapsing thrombotic thrombocytopenic purpura. All patients had elevated levels of von Willebrand factor. Although the clinical presentations of thrombotic thrombocytopenic purpura and severe preeclampsia with thrombocytopenia have many similarities, the underlying pathophysiology of each disorder appears to be different.

A specially designed rotational viscometer was used to investigate the effects of the antiplatelet agent PGI2 in combination with theophylline on the response of human platelets subjected to shear stress. Samples of citrated platelet-rich plasma (PRP) were exposed to shear stress in the viscometer for a period of 5 min at 23 degrees C. The levels of stress studied ranged from 50 to 300 dynes/sq cm. Pretreatment of the platelets with 0.01 microM PGI2 and 500 microM theophylline before exposure to shear stress caused a large reduction in shear-induced platelet aggregation. However, it was also observed that the PGI2-- theophylline pretreatment concomitantly caused a large increase in shear-induced platelet lysis and serotonin release at stress levels equal to or greater than 150 dynes/sq cm. This observed increase in platelet fragility may have important implications for clinical applications of PGI2. The results are discussed and compared to those obtained in prior work in which platelets were pretreated with acetylsalicylic acid or with PGE1.

Plasma VIII:von Willebrand factor antigen (VIII:vWF) levels were elevated approximately two- to eightfold in seven patients (three adults and four children) during acute episodes of thrombocytopenia, renal failure, and hemolytic anemia (the hemolytic-uremic syndrome, HUS). In all seven patients, there was an alteration in plasma VIII:vWF patterns during these acute HUS episodes, so that the largest VIII:vWF forms were relatively decreased. Plasma VIII:vWF multimer patterns returned to normal, or nearly to normal, as platelet counts returned to preexisting levels, even in the patients whose recovery of renal function was incomplete and whose plasma VIII:vWF antigen level remained above normal. The sister of one of the HUS patients had a similar clinical prodrome (gastroenteritis) that was not followed by thrombocytopenia or renal failure and was not accompanied by an elevated level or abnormal forms of plasma VIII:vWF. These results suggest that an alteration in VIII:vWF metabolism, distribution, or interaction with platelets is associated with acute HUS episodes. In contrast to patients with chronic relapsing thrombotic thrombocytopenic purpura, none of the HUS patients (either during or after the acute HUS episodes) had a defect in the conversion of unusually large VIII:vWF multimers derived from endothelial cells to the VIII:vWF forms found in normal plasma.

von Willebrand factor (vWF) multimers were examined in fetal, umbilical cord, and neonatal platelet-poor plasma (PPP) specimens. Sixty-five of 65 (100%) fetal PPP samples aged less than 35 weeks and seven of ten (70%) fetal samples aged greater than 35 weeks had unusually large vWF (ULvWF) multimers. Thirty of 46 (65%) cord PPP samples from neonates ranging in gestational age from 34 to 41 weeks had ULvWF. There was no significant relationship between either gestational age at time of delivery or birth weight and likelihood of finding ULvWF multimers in cord PPP samples. No maternal PPP sample contained ULvWF multimers. Serial heelstick samples from 16 preterm and term neonates were analyzed for 8 weeks. ULvWF multimers disappeared from the PPP of ten of the neonates during this time. The PPP of four neonates had vWF patterns similar to those in normal adult PPP throughout the sampling period. The ULvWF multimeric forms of fetal and neonatal PPP samples were similar to those constitutively released from endothelial cells. They were not as slowly migrating in a very porous 0.5% agarose gel system as the ULvWF multimers released from Weibel-Palade bodies in response to the calcium ionophore A23187. A vWF protomer was present in 97% of fetal samples, 83% of cord blood specimens, and 11% of neonatal heelstick samples, but was not found in any maternal sample. These results indicate that control mechanisms operative in older children and adults to prevent circulation of ULvWF multimers and vWF protomeric forms are normally acquired late in uterine life or during the neonatal period. ULvWF multimers, which are normal components of fetal, most cord, and some neonatal plasma samples, may contribute to in utero and postnatal hemostasis.

Unilateral hyphema, hematuria, and ecchymoses developed in a previously healthy 42-year-old women after the ingestion of aspirin and ethanol. There was no evidence for ocular trauma, disease, or vascular malformation by slit-lamp examination and gonioscopy. Platelet count and coagulation tests were normal. The patient's bleeding time was prolonged and there was impaired platelet aggregation. Delayed (secondary) aggregation in response to collagen, adenosine diphosphate, and epinephrine was decreased, as was aggregation induced by thrombin and serotonin. These data indicate that the qualitative platelet defect was induced by both aspirin and ethanol. Anterior chamber hemorrhage subsided after discontinuation of aspirin and ethanol, and the hyphema subsequently resolved. Bleeding time and platelet aggregation were normal two weeks after the patient's initial presentation. A prolonged bleeding time in association with normal platelet count, prothrombin time, and partial thromboplastin time indicated a qualitative platelet defect, which is most commonly drug-induced. Defective platelet function resulted in spontaneous hyphema.

A 72-yr-old male with a lifelong history of easy bruisability and posttraumatic bleeding had a prolonged prothrombin time and activated partial thromboplastin time. His plasma Stypven, Taipan, and Echis carinatus venom clotting times were prolonged. The presence of a dysprothrombin was confirmed by the discrepancy between plasma prothrombin coagulant activity and prothrombin antigen levels. His plasma prothrombin was capable of being completely absorbed onto and then eluted from barium sulfate. Crossed immunoelectrophoresis of his plasma prothrombin, and normal plasma prothrombin, into agarose containing rabbit anti-human factor II antibody were similar. Crossed immunoelectrofocusing, a procedure combining isoelectric focusing in disc gels with electroimmunoassay in the second dimension, demonstrated that the patient's prothrombin antigen was more basic than normal. The eluate from barium sulfate absorbtion of patient plasma, when reacted with Echis carinatus venom (which directly cleaves prothrombin to thrombin) clotted purified fibrinogen at a rate slower than normal plasma eluate. SDS-slab gel electrophoresis revealed that the prothrombin present in the patient's eluate was cleaved by Echis carinatus venom. These studies suggest that the coagulopathy of prothrombin Houston results from the generation of a dysfunctional thrombin.

The preparation and properties of a Mg2+-dependent, (Na+ + K+)-stimulated ATPase from platelets are described. The enzym system require Mg2+ for activity, is stimulated maximally by the presence of Na+ and K+ in a ratio of 11.5/1, has a pH optimum 7.30–7.40, and is half-maximally inhibited by ouabain at a concentration of 0.15 μM. The Km for ATP is approx. 0.4 mM. Enzyme activity is localized in both the “membrane” and “membrane-granule” subcellular fractions. Mg2+-dependent, (Na+ + K+)-stimulated ATPase activity is inhibited by ADP (which induces platelet aggregation) and by Pi. The inhibition by ADP is apparently not competitive with respect to ATP. Mg2+-dependent, K+-stimulated p- nitrophenyl phosphatase activity (a model for the externally-oriented K+-phosphatase portion of Mg2+-dependent, (Na+ + K+)-stimulated ATPase) is also inhibited by ADP. GDP, which does not induce aggregation of platelets, has no effect on the Mg2+-dependent, (Na+ + K+)-stimulated ATPase activity. It is postulated that ADP interacts with the external platelet membrane surface to inhibit Mg2+-dependent, (Na+ + K+)-stimulated ATPase activity with a resultant decrease in cation transport and membrane potential. This phenomenon may play a role in platelet aggregation.

A 48-year-old black woman developed severe bilateral hypoxic proliferative retinopathy without other clinical manifestations. The hemoglobin level was 10.6 to 11.5 g/100 ml, reticulocyte level was 2.2%, targeted and sickled red blood cells were seen on blood smears, and hemoglobins S and C were demonstrated by electrophoresis. Glucose tolerance test was normal. The development of neovascular proliferation, vitreous hemorrhage, and retinal detachment unassociated with other clinical symptoms is unusual in sickle cell hemoglobin C disease.

Cytochalasin B alters the structure and functional properties of filamentous actin. Platelet-mediated clot retraction in dilute platelet-rich plasma (PRP) is inhibited progressively at cytochalasin B concentrations of 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml. Dynamic rheological measurements of recalcified PRP in a Weissenberg Rheogoniometer indicate that platelet contractility (as reflected in measurements of elastic moduli) is reduced by 33%, 57% and 63% at cytochalasin B concentrations of 0.01, 0.05 and 0.1 mg/ml, respectively. In contrast, pre-incubation of human platelet-rich plasma (PRP) with 0.01 mg/ml or 0.05 mg/ml cytochalasin B does not inhibit collagen-induced [14C]serotonin release on collagen-induced-platelet aggregation, which is dependent on the release of ADP from platelet dense granules. Even at a cytochalasin B concentration of 0.1 mg/ml, collagen-induced [14C-]serotonin release and aggregation are impaired only moderately. Cytochalasin B does not interfere with the uptake by platelets of [14C-]-serotonin, or with the kinetics and extent of clot formation in platelet-free plasma. Thus, concentrations of cytochalasin B which impair platelet contractility do not inhibit the release of platelet dense granule contents. It is concluded that neither the platelet release reaction nor platelet aggregation is dependent on platelet contractile mechanisms.

Tumor cells in primary cultures derived from 11 untreated nonhemopoietic cancer patients were reacted with specifically coated sheep erythrocytes. Rosette formation between tumor and indicator cells was assessed, Eight of the primary cultures reacted positively with both IgG-coated (EAIgG) and with IgM-human complement coated (EAIgMC or EAIgMC 4,3) sheep erythrocytes. EAIgG rosette formation in positive cultures ranged from 25 to 85%, and for EAIgMC/EAIgMC 4,3 reactivity ranged between 22--95%. Rosette formation with E (uncoated) and EAIgM was negligible. These findings suggest that human nonhemopoietic tumor cells may carry on their surface receptor sites for an IgG component of immune complexes and for human complement. These receptor sites may be important in the host-tumor relationship.

The effect of dialyzer membrane and design on hemostatic parameters during hemodialysis were evaluated in a prospective controlled study. This study demonstrated that hemodialysis is associated with significant platelet activation and loss, which are influenced by both dialyzer configuration and membrane composition. In addition, use of the cuprophan membrane is associated with greater perturbations of the vascular endothelium, as reflected in changes in factor VIII-related von Willebrand factor and 6-keto-prostaglandin F1 alpha concentrations not seen with the polyacrylonitrile membrane. Of the dialyzers studied, the polyacrylonitrile membrane in a hollow-fiber configuration appears to minimize platelet loss and activation, and to minimize increases in factor VIII-related von Willebrand factor and 6-keto-prostaglandin F1 alpha.

Thrombotic thrombocytopenic purpura (TTP) and the haemolytic-uraemic syndrome (HUS) are caused by platelet thrombi in the microcirculation (i.e. arterioles and capillaries) throughout the body (TTP) or predominantly in the kidneys (HUS). Plasma factors that induce intravascular platelet agglutination have been a focus of investigation into the pathogenesis of these disorders. Von Willebrand factor (vWF) multimeric forms that are larger than those present in normal plasma are found in the plasma of patients with the chronic relapsing form of TTP. These unusually large vWF multimers are similar to those produced by normal human endothelial cells, but never allowed into the normal circulation. Unusually large vWF multimers in chronic relapsing TTP patients are most apparent in plasma during remission. They disappear, presumably in the process of attaching to platelets and inducing the formation of platelet thrombi, during relapses in chronic TTP. The disappearance of the largest plasma vWF multimeric forms during acute episodes of non-relapsing TTP and HUS has also been seen. These syndromes may be the result of damage to systemic or renal endothelial cells. A cofactor which induces the attachment of large vWF multimers to platelets during episodes of TTP has recently been detected, but not yet characterized biochemically. The cryosupernatant (i.e. vWF-depleted) fraction of normal plasma contains an activity that converts, or potentiates the conversion of, unusually large vWF multimers to the somewhat smaller circulating vWF forms as the bloodstream. There is clinical evidence that an autoantibody may prevent the effect of this 'unusually large vWF depolymerase' in some chronic relapsing TTP patients. Transfusions of normal plasma or cryosupernatant as prophylaxis against, or therapy for, episodes of TTP may transiently provide this missing unusually large vWF depolymerase activity, as well as additional plasma proteins to bind and eliminate the vWF cofactor proposed as the inciting agent of TTP episodes. In some patients, partial removal of unusually large vWF multimers (and possibly the inciting vWF cofactor) by plasmapheresis may be required along with the transfusion of normal plasma or cryosupernatant, in order to control in vivo platelet agglutination. Plasma manipulation has greatly improved the survival of patients with relapsing and non-relapsing forms of TTP. Corticosteroids may also be beneficial. The effectiveness of ancillary measures (splenectomy, vinca alkaloids or other immunosuppressive drugs) is not precisely defined. There is no convincing evidence that aspirin, dipyridamole or PGI2 are helpful in TTP.(ABSTRACT TRUNCATED AT 400 WORDS)

Sudden thrombocytopenia that was temporally related to the administration of morphine sulfate developed in a 23-year-old woman. A morphine-dependent platelet antibody was found in her serum by chromic chloride Cr 51 platelet lysis. The antibody was complement dependent, present in the IgG immunoglobulin fraction, and its drug-dependent platelet lytic activity was demonstrable with several narcotic analgesics in addition to morphine. The antibody activity declined over an eight-month period following recovery from thrombocytopenia. To our knowledge, morphine-induced immune thrombocytopenia has not previously been described.

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.

Ristocetin induces platelet agglutination in the presence of human factor VIII-associated ristocetin cofactor (vWF). The specificity, extent, and tenacity of binding among these reactants during agglutination and deagglutination were examined. Purified human vWF polymers were radioiodinated and reisolated. Radioiodinated vWF, a disulfide-linked polymer of 230,000 dalton subunits, attached to formalinized human platelets only in the presence of ristocetin. This binding reached equilibrium within 30 sec, and as ristocetin concentrations were raised from 0.2 mg/ml, the extent of attachment increased progressively to reach maximum at 0.5 to 0.6 mg/ml ristocetin. Ristocetin-induced binding was inhibited by vancomycin, unlabeled-purified vWF polymers, normal and hemophilia A plasma, and rabbit anti-human vWF. Binding was not impaired by plasma without detectable vWF or by naturally occurring human IgG antibodies to factor VIII coagulant activity. When formalinized platelets were pelleted from suspensions containing 125I-ristocetin, small quantities of radiolabeled ristocetin associated with platelets both in the presence or absence of vWF. About 95% of the attached 125I-ristocetin was removed by subsequent washes in buffered saline. The attachment of unmodified ristocetin or 125I-ristocetin to platelets, or the formation of complexes with vWF, could not be detected by agarose column chromatography, sucrose cushion ultracentrifugation, or equilibrium dialysis. These results indicate that (1) the initial binding of human vWF polymers to platelets is a specific interaction which requires the presence of ristocetin; (2) ristocetin and human vWF do not form persistent complexes in solution; and (3) the association of ristocetin and platelets is of low affinity.

A rotational viscometer was used to study the effects of shear stress on platelets in human platelet-rich plasma (PRP). For 5-min exposure times, shear stresses above 160 dynes/cm2 induced platelet lysis (as determined by release of platelet lactic dehydrogenase). For 30-s exposure times, shear stresses greater than 600 dynes/cm2 were required to induce platelet lysis. The platelet counts of sheared PRP were decreased to as low as one-fifth the original count due largely to shear-induced aggregation. The count is a minimum at intermediate stress levels (200-400 dynes/cm2). Higher stresses induce disaggregation as well as lysis. The diminution in the counts was partially reversed in 2 h incubation after cessation of shearing. Experiments were carried out with three different viscometer configurations so that the shear stress and the solid surface area access could be varied independently. Surface access was not a significant variable in the conditions of the experiments. Thus aggregation and lysis may be induced by stress effects alone as well as by solid surface effects. The results also show that the response of platelets to shear stress is strongly dependent on exposure time. Platelets are much less resistant to shear stress than red cells for relatively long exposure times. However, the converse is true for very short exposure times.

Serum complement levels were assayed in 26 patients with disseminated cancer, who received immunotherapy with infusion of C. parvum. Complement activation, indicated by the consumption of C3 or C4 or both, was found in 46% of the patients. Serum samples showed direct correlation between decreased C3 and conversion of C3 proactivator, whereas such conversion did not occur when C4 alone was decreased. It is concluded that the bypass (properdin) pathway was activated in patients in whom C3 consumption was detected, while the classical (C1) pathway was activated in the patients with C4 consumption unaccompanied by C3 decrease. Direct correlation was observed between delayed cutaneous hypersensitivity reactions to recall antigens and the incidence of C. parvum-associated complement activation.

Depending on the time of addition, prostaglandin I2 (PGI2; greater than or equal to 10(-9) M) either inhibits or reverses platelet agglutination mediated by human factor VIII-related von Willebrand factor activity (FVIIIvWF) and ristocetin, or bovine FVIIIvWF alone. 6-Keto-PGF1 alpha, the inactive metabolite of PGI2, is without effect, PGI2 inhibition is potentiated by the phosphodiesterase inhibitor, theophylline, and is not the result of PGI2 suppression of ADP release. PGI2 (+/- theophylline) does not inhibit ristocetin-induced binding of purified human 125I-FVIIIvWF multimers to washed platelets or to platelets treated with PGI2 and then formalin fixed (although subsequent agglutination of these platelets is impaired). Washed platelets treated previously with 2-aminoethylisothiouronium bromide (AET), an agent that reduces disulfide bonds and alters platelet membranes, also bind human 125I-FVIIIvWF multimers without agglutinating. We conclude that FVIIIvWF-mediated agglutination requires both functional platelet FVIIIvWF binding sites and platelet-platelet cohesion sites, and that platelet surface cohesion sites are altered by AET and PGI2. PGI2 from adjacent intact endothelial cells may prevent excessive platelet accumulation on exposed subendothelium without suppressing an essential hemostatic process--the binding of platelets to subendothelial FVIIIvWF.

In dilute suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP), dibutyryl-cAMP (DBcAMP) and monobutyryl-cAMP inhibited platelet-mediated fibrin clot retraction in concentrations of 2--3 X 10(-6) M, with complete inhibition at 1--3 X 10(-4) M. Prostaglandin E1 (PGE1), which inhibited fibrin clot retraction in concentrations greater than 1.5--3 X 10(-8) M, was a more effective inhibitor than either PGE2 or PGF2 alpha. In the presence of theophylline (10-4 M), concentrations of DBcAMP, PGE1, PGE2 and PGF2 alpha necessary to inhibit fibrin clot retraction were reduced 50-fold for DBcAMP and 2.5 to 20-fold for the prostaglandins. In dilute PRP or GSP, inhibition of fibrin clot retraction does not result from inhibition of thrombin-induced platelet aggregation. Thus, compounds which increase platelet cAMP levels result in the inhibition of platelet-mediated fibrin clot retraction, and this inhibitory effect may be mediated, at least in part, through suppression of platelet contractility. Cyclic GMP, dibutyryl-cGMP and carbamylcholine-Cl (which stimulate guanylate cyclase) did not influence fibrin clot retraction, and did not prevent inhibition of fibrin clot retraction by DBcAMP and PGE1. Colchicine, in concentrations known to disrupt platelet microtubules (2.5 X 10(-6) M to 2.5 X 10(-3) M), had little inhibitory effect on either fibrin clot retraction or platelet (3H)-serotonin release.

A 70 year old Black woman had chronic hemolytic anemia without recurrent painful crises. Hemoglobin pattern by electrophoresis was hemoglobin S (69 to 71 per cent), hemoglobin A2 (4.6 per cent) and hemoglobin F (24 to 27 per cent). No hemoglobin A was detected, and the hemoglobin F was distributed heterogeneously in the red cells. Reticulocyte alpha/nonalpha globin chain synthetic ratios were 1.44 to 1.62. Thus, the patient had a high hemoglobin F variant of S-beta zero (betao) thalassemia which has not been described previously. Her clinical course has been mild in comparison with S-betao thalassemia patients who do not have extremely elevated hemoglobin F levels.

To analyze and review von Willebrand factor (vWF) multimeric patterns in patients with single-episode thrombotic thrombocytopenic purpura (TTP), intermittent TTP (episodes at infrequent, irregular intervals), chronic relapsing TTP (episodes at frequent, regular intervals), and the hemolytic-uremic syndrome (HUS).Platelet-poor plasma samples were obtained in EDTA, citrate, or citrate-hirudin-aprotinin-leupeptin from 36 patients with single-episode TTP, eight patients with intermittent TTP, four patients with chronic relapsing TTP, and 26 patients with HUS. The samples were separated by sodium dodecyl sulfate-agarose gel electrophoresis, overlaid with rabbit 125I-anti-human vWF IgG, and analyzed by autoradiography.Abnormalities of vWF multimers were found in platelet-poor plasma samples from 31 of 36 found in platelet-poor plasma samples from 31 of 36 patients (86%) at the onset of and during their single TTP episode. vWF multimers larger than those in normal plasma, and similar to vWF forms observed within normal human endothelial cells (unusually large vWF multimers), were demonstrated in 31% of the patients; 19% had either unusually large vWF multimers or a relative decrease in the largest plasma vWF forms in different serial samples; 36% had a relative decrease in the largest plasma vWF forms. These results imply that endothelial cell injury or intense stimulation, along with the attachment of unusually large vWF multimers and the largest plasma vWF forms to platelets, occurred during the single TTP episodes in most patients. Patterns of vWF multimers were normal in 92% of patients with single-episode TTP studied after recovery. All eight patients with intermittent TTP and the four patients with chronic relapsing TTP had unusually large vWF multimers in their plasma between episodes, and these multimers decreased or disappeared during relapses. Of 26 children and adults with HUS, 14 had a relative decrease in the largest plasma vWF multimeric forms and one had unusually large vWF multimers during the episode (vWF multimeric abnormalities in 58% of the patients).It is probable that vWF was involved in the pathophysiology of TTP in most of these patients with the single-episode, intermittent, or chronic relapsing types of TTP, and in more than 50% of the patients with HUS.

Suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP) can be used to evaluate clot retraction subsequent to platelet aggregation and fibrin formation. PRP (200,000 per cubic millimeter) or GSP (200,000 or 100,000 per cubic millimeter) are diluted 1:10 (PRP) or 1:8 (GSP) in phosphate buffer, pH 7.4, and clotted with a high concentration (2.5 U. per milliliter) of thrombin. Human fibrinogen (25 mg. per cent) is added to GSP prior to dilution. Clot retraction is 91 to 100 per cent completed in 1 hour and is quantified by measurement of residual fluid volume. Test conditions are unfavorable for fibrinolysis. Very low concentrations of fibrin/fibrinogen degradation products D and E are detected in residual fluid, and no erythrocyte fall-out occurs. Furthermore, the extent of retraction in the dilute systems is related only to platelet numbers and platelet function. The dilute PRP and GSP methods allow evaluation of clot retraction in the presence of PGE1, the most potent inhibitor of platelet aggregation induced by conventional concentrations of collagen, ADP, epinephrine, and thrombin (0.1 to 0.5 U. per milliliter). High concentrations of PGE1 (to 6 x 10(-6) M) do not inhibit aggregation of GSP, fibrin formation, or platelet-fibrin interaction induced by 2.5 U. per milliliter of thrombin. In contrast, PGE1 concentrations as low as 1.5 to 3.0 x 10(-8) M inhibit clot retraction in both the dilute PRP and GSP systems. Thus, using dilute PRP or GSP the effects of platelet aggregation inhibitors on clot retraction can be determined independently of effects on platelet aggregation.

Excerpt Cavernous transformation of the portal vein may be a sequela of presinusoidal portal vein obstruction as small veins dilate in a compensatory effort to provide circulation to the liver (1, ...