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Leukocyte-derived microparticles (MPs) are markers of cardiovascular diseases and contribute to pathogenesis by their interaction with various cell types. The presence and activation state of a multifunctional leukocyte receptor, integrin αMβ2(CD11b/18), on MPs derived from human neutrophils (PMNs) were examined. αMβ2expression was significantly enhanced on MPs derived from stimulated compared with resting PMNs. Furthermore, αMβ2on MPs from stimulated but not resting PMNs was in an activated conformation because it was capable of binding activation-specific monoclonal antibodies (CBRM1/5 and mAb24) and soluble fibrinogen. MPs expressing active αMβ2interacted with and were potent activators of resting platelets as assessed by induction of P-selectin expression and activation of αIIbβ3. With the use of function-blocking antibodies and MPs obtained from αM−/−-deficient mice, we found that engagement of GPIbα on platelets by αMβ2on MPs plays a pivotal role in MP binding. Platelet activation by MPs occurs by a pathway dependent on Akt phosphorylation. PSGL-1/P-selectin interaction also is involved in the conjugation of MPs to platelets, and the combination of blocking reagents to both αMβ2/GPIbα and to PSGL-1/P-selectin completely abrogates MP-induced platelet activation. Thus, cooperation of these 2 receptor/counterreceptor systems regulates the prothrombotic properties of PMN-derived MPs.

Previous studies demonstrated that integrin αMβ2(CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing αMβ2, uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that αMβ2supported adhesion and migration of these cells to uPA, and, in each case, blockade of αMβ2suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by αMβ2, which are distinct from the growth factor domain that binds to uPAR. Within the αMsubunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and αMβ2, both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of αMβ2reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by αMβ2. Thus, recognition of uPA by αMβ2allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and αMβ2. This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.

Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IlIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by >85%. T10, an antibody specific for the GPIIb—IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.

Human plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) were radioiodinated and their interaction with washed human platelets was assessed. Both ligands were bound by the platelet at 37 °C, and an apparent equilibrium was attained within two hours. Minimal binding was observed at 22 °C or 4 °C. The specificity of these interactions was indicated by the observations that: (a) labeled and nonlabeled lipoproteins interacted with the platelet with the same apparent affinity; (b) nonlabeled lipoproteins inhibited binding, whereas unrelated plasma proteins did not; and (c) the platelet-bound ligands exhibited the appropriate apoprotein chain compositions when analyzed by polyacrylamide gel electrophoresis. Binding of HDL and LDL was found to be independent of the state of platelet activation and did not require divalent ions. Binding of HDL to the platelet was saturable, and a class of sites that maximally bound 1,585 ± 390 HDL particles, with a dissociation constant of 3.1 × 10–8mol/L, was identified. Binding of LDL to the platelet was more complex, but evidence for a class of sites that bound 7,075 ± 4,800 LDL particles, with a dissociation constant of 4 × 10–8mol/L, was found. LDL was a poor inhibitor of 125I-HDL binding to the platelet, whereas HDL was an effective inhibitor of 125I-LDL binding. The capacity of HDL to bind or inhibit LDL binding was not dependent on its apoprotein E content. These results are most readily interpreted in terms of two types of lipoprotein interaction sites on platelets: (1) an HDL binding site that does not bind or interacts poorly with LDL, and (2) an LDL binding site that recognizes or is otherwise altered by HDL. The HDL site may be similar to the HDL receptor expressed by steroidogenic tissues in terms of apoprotein specificity. The LDL site is not the same as the LDL receptor of most extrahepatic cells.

Endothelial cell receptors for tissue plasminogen activator (t-PA) have been demonstrated recently, and we have sought to identify a region of the t-PA molecule involved in its interaction with these receptors on human umbilical vein endothelial cells. Of three monoclonal antibodies against various regions of t-PA, one directed against the finger region inhibited 125I-t-PA binding to the cells. Synthetic peptides corresponding in amino acid sequences to segments from within the finger region were constructed, and one of these inhibited t-PA binding. This peptide corresponded to residues 7 through 17 of t-PA. The inhibition by this peptide was specific as other peptides from the finger region were inactive. The inhibitory peptide also did not affect the binding of another fibrinolytic ligand, urokinase, to the cells. Although a role for other regions of t-PA in binding to endothelial cells cannot be excluded, the results implicate a short span of linear amino acid sequence within the finger region in the interaction of t-PA with endothelial cells.

An antigen immunochemically indistinguishable from plasma α2-antiplasmin, the primary plasmin inhibitor, was detected in human platelets. By radioimmunoassay, 33–114 ng α2-antiplasmin antigen was quantitated in the detergent-soluble extract of 109washed human platelets from 10 normal donors with a mean level of 62 ± 24 ng/109platelets. Plasma α2-antiplasmin, either in the platelet suspending medium or on the surface of the platelets, could account for less than 8% of the antigen present in the platelet extracts. When stimulated with thrombin, the platelets released α2-antiplasmin antigen without cell lysis, and >85% of the α2-antiplasmin antigen was released at a high thrombin dose. At a lower dose of thrombin, α2-antiplasmin and platelet factor 4 were partially released without concomitant secretion of serotonin. No α2-antiplasmin antigen was detected in extracts of red blood cells, polymorphonuclear leukocytes, and adherent and nonadherent mononuclear cells. Thus, the platelet is the only peripheral blood cell containing significant amounts of α2-antiplasmin.

To analyze molecular mechanisms of platelet aggregation, we have studied the aggregation of Chinese hamster ovary (CHO) cells expressing between 1 and 4 × 105recombinant human glycoprotein (GP) llb-lll, molecules per cell (A5 cells). These cells aggregated as measured by the disappearance of single cells during rotary agitation. Aggregation was dependent on the presence of extracellular fibrinogen ( = 500 nmol/L) and divalent cations, and required prior activation of the GPIIb-llla. A synthetic peptide (GRGDSP) and monoclonal anti-GPIIb-lllaantibody (2G12) that block platelet aggregation also blocked aggregation of these cells. Parent CHO cells or those expressing recombinant GPIIb-lllacontaining a point mutation that causes variant thrombasthenia both failed to aggregate when stimulated in the presence of fibrinogen. These data show that GPIIb-lllais the only unique platelet surface component required for aggregation.

Plasma fibronectin binds in a specific and saturable manner to thrombin-stimulated platelets. 7-Thrombin stimulated 80% as much fibronectin binding to platelets as a-thrombin with conversion of ≤1% of platelet fibrinogen to fibrin. Afibrinogenemic and normal platelets bound similar quantities of fibronectin in the presence of calcium or magne-sium-ethylene glycol tetra-acetic acid (EGTA). These observations indicate that fibronectin can interact with platelets without involvement of fibrin or fibrinogen. Nevertheless, two different effects of fibrin(ogen) on fibronec-tin binding were observed. First, exogenous fibrinogen inhibited fibronectin binding to thrombin-stimulated platelets. This inhibition was unidirectional, as fibronectin did not inhibit fibrinogen binding to ADP or thrombin-stimu-lated cells. Second, formaldehyde-fixed cells with surface-associated fibrin bound significant quantities of fibronectin. This interaction required calcium and did not occur on fixed cells with or without surface-bound fibrinogen. A portion of the ligand bound to fixed cells with surface-associated fibrin was modified to form a derivative with a molecular weight identical to that of the fibronectin subunit crosslinked to the a-chain of fibrin. This high mol wt derivative was also observed to a variable extent with living cells in the presence of magnesium or calcium but not in the presence of magnesium-EGTA. Thus, fibronectin binds to platelets by at least two mechanisms: (1) a fibrin(ogen)-independent pathway that requires divalent ions and is inhibited by exogenous fibrinogen; and (2) a fibrin-depen-dent pathway with an absolute calcium requirement. With nonaggregated, thrombin-stimulated platelets, the former pathway appears to predominate. © 1985 by Grune & Stratton, Inc.

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 μmol/L) and high capacity (0.71 to 3.3 x 106receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 μmol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 106sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 μmol/L and 1.1 x 106sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inacti-vated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50, (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125l-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.

Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPMb (PMI-1) or Ilia (anti-LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGOSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenia variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to Increase the binding of PMI-1 or anti-LIBS1 to the thrombasthenia platelets, suggesting a primary defect In ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient’s GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate–stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

A deficiency of α2-antiplasmin has been identified in a female patient with severe and frequent bleeding episodes. Routine coagulation and platelet assays of the patient's plasma were within normal limits. However, abnormally rapid whole blood or dilute plasma clot lysis times and an abnormal FXIII test in which clots were lysed in the presence of urea or saline suggested an abnormal fibrinolytic system. Analysis of α2-antiplasmin levels by radioimmunoassay revealed <1.0 μg/ml α2-antiplasmin. Functional assays indicated an α2-antiplasmin level <10% of normal. Addition of purified α2-antiplasmin to the patient's plasma restored its ability to inhibit plasmin in in vitro assays, and mixtures of patient plasma with normal plasma did not interfere with the antiplasmin activity of the normal plasma.Whereas normal platelets contain 68 ng α2-antiplasmin/109platelets, platelets from the patient contained 30% of the normal level of antigen. Analysis of α2-antiplasmin functional and antigenic levels in the plasma of both parents and four siblings of the propositus provided evidence consistent with an autosomal mechanism of inheritance of α2-antiplasmin deficiency. One sibling appeared to be homozygous and three siblings and the parents were heterozygous for the deficiency. Two heterozygotes had positive bleeding histories. The association of a bleeding disorder with a deficiency of α2-antiplasmin emphasizes that lack of regulation of the fibrinolytic system can result in a hemostatic dysfunction.

Platelet membrane GPIIb is comprised of a disulfide-linked heavy chain (GPIIb(H)) and light chain (GPIIb(L)). We have examined the role of the two chains of GPIIb in the maintenance of the GPIIb-llla heterodimer and Arg-Gly-Asp (RGD) peptide-binding function. Lysates of surface radioio-dinated platelets were treated with 1 % 2-mercaptoethanol for 18 hours at 4°C. Reduction of the interchain disulfide in GPIIb was followed by immunoprecipitation with antipep-tide antibodies specific for GPIIb(H) or GPIIb(L). In addition to the GPIIb-llla complex, a polypeptide of 120 Kd was precipitated by anti-GPIIb(H) and a polypeptide of 23 Kd was precipitated by anti-GPIIb(L) from reduced platelet lysates. To determine whether GPIIb(H) or GPIIb(L) remained complexed with GPIIIa, reduced platelet lysates were immunoprecipitated with AP3, a monoclonal anti-GPIIIa antibody, resulting in the coimmunoprecipitation of GPIIb(H) but not GPIIb(L). Conversely, the monoclonal anti-GPIIb(H) antibody PMI-1 immunoprecipitated GPIIIa with GPIIb(H). Thus GPIIb(H) maintains its association with GPIIIa. Furthermore, the GPIIb(H)-llla complex retains its reactivity with AP2, a monoclonal antibody (MoAb) specific for the nondissociated GPIIb-llla complex. Affinity chromatography of reduced platelet lysates on immobilized KYGRGDS resulted in binding and specific elution of the GPIIb(H)-llla complex. These findings indicate that GPIIb(H) contains sufficient information for maintenance of a complex with GPIIIa and support of the binding of the heterodimer to RGD peptides.

Fibrinogen binds to specific receptors on human washed platelets and these sites are induced by adenosine diphosphate (ADP). This interaction is assumed to be the basis for the participation of the molecule in ADP-stimulated aggregation of platelets, but fibrinogen binding to platelets in plasma has not been directly demonstrated. In this study, we have characterized the interaction of 125l-fibrinogen to platelets in the platelet-rich plasma (PRP) of afibrinogenemia patients. In either citrated or heparinized PRP, association of fibrinogen with platelets was demonstrable and was dependent on ADP dose. This binding reached equilibrium in 10–15 min, and saturation was achieved at fibrinogen concentrations greater than 0.5 μM.A linear Scatchard plot was derived that indicated a single class of binding sites with an affinity constant of Ka= 1.8 x 106M-1and 32.000 fibrinogen molecules were maximally bound per platelet. The kinetics of the platelet fibrinogen interaction in plasma were essentially the same at 37°C and 22°C. but fewer molecules were bound at 37°C. The rate constants of association were kon22°C= 0.9 x 106M-1·min-1and kon37°C= 0.4 x 106M-1· min-1, respectively. Stabilization of the platelet-bound fibrinogen occurred only to a partial extent in both heparinized and citrated plasma. These results are similar to those obtained with washed platelets and establish that the previously defined steps in ADP-induced binding of fibrinogen to platelets occur in plasma, namely receptor induction by ADP, initial reversible binding, and irreversible binding.

The monoclonal antibody, OKM5, recognizes an 88-Kd monocyte membrane protein and also binds to the platelet membrane protein, GPIV (GPIIIb, CD36). In this study, we have found that the OKM5 target epitope is present at approximately 12,000 copies per platelet and that interaction with the antibody has both stimulatory and inhibitory effects on platelet function. In the absence of other stimuli, OKM5 induced platelet aggregation, secretion, and expression of fibrinogen receptors. These stimulatory responses required intact antibody as F(ab’)2fragments were not active but blocked the stimulatory activity of the intact antibody. In contrast, exposure of platelets to OKM5 followed by another strong stimulus such as thrombin resulted in a marked suppression of fibrinogen, fibronectin, and von Willebrand factor binding to the cells. This effect was not noted when a weak stimulus, adenosine diphosphate, was the second agonist. At OKM5 concentrations that interfered with fibrinogen binding to thrombin-stimulated platelets by 80% to 90%, platelet binding of exogenous thrombospondin, or surface expression of endogenous thrombospondin was not affected. The inhibitory effect of OKM5 on fibrinogen binding to thrombin-stimulated platelets was related to the formation of massive platelet aggregates in the samples. These results show that interaction of OKM5 with its target antigen on platelets can elicit diverse functional responses from the cells.

Thrombospondin (TSP), a platelet a-granule protein, becomes expressed on the surface of thrombin-stimulated platelets. The surface expression of this protein occurs through two distinct mechanisms. At low platelet concen trations (1 × 108/mL), a divalent ion-independent, low-capacity mechanism predominates. At higher cell con centrations, a divalent ion-dependent, higher capacity mechanism prevails that can account for >90% of all the TSP surface expression measured. This mechanism requires the presence of both calcium and magnesium (Ca + Mg). The dependence of the divalent ion-dependent surface expression on platelet concentration suggests that release of the molecule from the cell followed by its binding to the cell surface mediates this component of the endoge nous TSP-platelet interaction. These data are consistent with a two-receptor model for the platelet surface expres sion of the endogenous TSP pool.

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab’ fragment) directed against the glycoprotein (GP) Ilb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25°C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP Ilb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to “strong” agonists exhibit an increased number of surface-oriented epitopes associated with GP Ilb/IIIa. The GP Ilb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.

Platelet membrane GPIIb-IIIa is a member of the integrin family of heterodimeric adhesion receptors. Processing and export of certain leukocyte and melanoma integrins is disrupted in cells lacking one subunit. We found that surface expression of GPIIb-IIIa, measured by fluorescent activated cell sorting or by surface labeling, required cotransfection of both subunits. In contrast, surface expression was not detected when the subunits were transfected individually. Immunoprecipitation of metaboli- cally labeled transfected cells confirmed the presence of comparable levels of intracellular protein in all cases. When both subunits were transfected, post-translational cleavage of Pro-GPIIb to yield GPIIb heavy chain was also seen, while transfection with GPIIb alone resulted in coprecipitation of Pro-GPIIb with a second band that may be an endogenous β subunit. Pro-GPIIb in these transfectants was not processed to yield GPIIb heavy chain. When transfected into COS cells alone, transiently expressed GPIIIa remained intracellular and did not appear to complex with any endogenous proteins. Thus, surface expression of processed GPIIb-IIIa depends on the presence of both subunits; the coordinate reduction of both subunits observed in some cases of Glanzmann's thrombasthenia may result from mutation affecting only one.

Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). Because calpains and one or more enzymes released from polymorphonuclear leukocytes are known to proteolyze von Willebrand factor (vWF) in vitro with resultant loss of large multimers similar to that seen in IIA vWD, they have been suggested as being responsible for the proteolysis in vivo. Using monoclonal epitope mapping, we have examined the proteolysis of the vWF subunit by porcine calcium-activated neutral proteases (calpains) and human leukocyte elastase to determine whether they produce the vWF proteolytic cleavage products seen in normal individuals and IIA vWD. Purified vWF was digested with porcine calpains I and II. We found no difference in the size, location, and quantity of the fragments produced by calpain I v calpain II. New fragments were detected of approximately 200,170,150, and 125 Kd. There was no evidence for generation of the native 140 and 176 Kd fragments. Some loss of the native fragments was seen, which suggests that they were further cleaved. Epitope mapping of the 170- and 150-Kd calpain-cleaved fragments revealed them to be from different parts of the molecule than the regions from which the native 176- and 140-Kd fragments derived. This was further supported by determination of the amino-terminal sequence of the calpain-cleaved 170- and 150-Kd fragments. Digestion of vWF with human leukocyte elastase produced new fragments at 210/205, 190, 170/165, 145/140, and 130/125 Kd. No generation of native fragments was detected. Monoclonal epitope mapping of the 145/140-Kd elastase-cleaved band proved that it derived from the carboxyl-terminal portion of the vWF molecule, whereas the native 140-Kd fragment is derived from the amino-terminal end. Neither calpains nor human leukocyte elastase produced the proteolyzed fragments present in normal and IIA vWD and, therefore, probably do not cause the loss of large multimers that is seen in that disorder. © 1988 by Grune & Stratton, Inc.

Endothelial cells are centrally involved in regulation of fibrinolysis, and receptors for plasminogen and urokinase provide a mechanism by which cells can regulate their fibrinolytic function. Therefore, the existence and characteristics of receptors for these fibrinolytic components on cultured human umbilical vein endothelial cells were examined. We verified the presence of plasminogen receptors on these cells (Kd= 2.1 ±1.3 μmol/L, and 1.8 ± 1.3 × 107binding sites/cell). These binding parameters and other characteristics indicate that these receptors are closely related to the plasminogen receptors on many circulating and adherent cells. Specific binding sites that interact with two-chain urokinase of mol wt 55,000 with a dissociation constant of 2.1 ± 1.7 nmol/L, with 2.9 ± 2.9 × 105sites/cell were also identified. Single-chain urokinase of mol wt 55,000, but not the two-chain degradation product of mol wt 33,000 bound to the cells, implicating the amino-terminal aspects of the ligand in receptor recognition. When endothelial cells were stimulated with thrombin, an agent that modulates their fibrinolytic potential, both receptor types were modestly affected; urokinase binding increased 17%, whereas plasminogen binding decreased 19%. The presence and modulation of plasminogen and urokinase receptors provide a potentially important additional mechanism by which endothelial cells may regulate fibrinolysis. © 1988 by Grune & Stratton, Inc.

Thrombin and adenosine diphosphate (ADP) supported the binding of 125I-fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.