Fibrinogen Binding Site Research Articles

Glycoprotein V hydrolysis by thrombin. Lack of correlation with secretion

Platelet membrane glycoprotein V (GPV) was hydrolyzed during thrombin-induced platelet aggregation releasing a fragment GPV f1 into the supernatant. Hydrolysis of GPV required catalytically active thrombin and was diminished by chemical modification of the fibrinogen binding site of thrombin. Half maximal liberation of GPV f1 occurred at a 10-fold higher concentration of thrombin than was required for half-maximal release. Time course studies at several thrombin concentrations showed disparate release of GPV f1 and thrombospondin. These results emphasize the complexity of the initial events in thrombin-induced platelet activation.

Monoclonal antibodies against human platelet membrane glycoproteins IIb-IIIa II. Different effects on platelet function

Preincubation of human platelets with two pairs of immunologically identical monoclonal antibodies (mab) directed against epitopes on the membrane glycoprotein IIb-IIIa complex - two of them (Gi3 and Gi5) precipitating glycoproteins IIb-IIIa, and two (Gi4 and Gi6) recognizing glycoprotein IIIa -induced slightly different inhibitory effects on platelet function. Gi3 and Gi5 blocked platelet aggregation and 14C-serotonin release from platelets induced by ADP, collagen, thrombin, arachidonic acid, ionophore A 23187, platelet activating factor and ristocetin (in the presence of divalent ions). Ristocetin-induced platelet agglutination (in the presence of EDTA) was not inhibited by these mab. Gi4 had no effect on platelet aggregation and release. Gi6 had the same effect on platelet function as Gi3 and Gi5, but only when incubated with Zw(a) (PIA1)-positive platelets with the exception that collagen-induced aggregation was not affected. We conclude that the similar, though not identical, effects on platelet function induced by mab against different epitopes on the glycoprotein complex IIb-IIIa indicate an “unspecific” interference with platelet membrane function possibly due to steric hindrance by mab of the fibrinogen receptor or by inhibition of conformational changes in the GP IIb-IIIa complex necessary for exposure of the fibrinogen binding site.

Identification and characterization of fragments of major glycoproteins from platelet membrane after chymotrypsin treatment

Human platelets were surface-labeled by the periodate/NaB3H4 method or by lactoperoxidase-catalysed iodination with 125I. The labeled platelets were treated with chymotrypsin under conditions known to give platelets which aggregate with fibrinogen without stimulation with ADP. Platelets and supernatant were then analysed by various gel electrophoretic techniques including isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions and two-dimensional non-reduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography or indirect autoradiography. Chymotrypsin-treatment of surface-labeled platelets degraded the major glycoproteins Ib, IIb and IIIa but also GP120(4.9-5.4), GPIc and GPV. The membrane-bound fragments of GPIb, IIb and IIIa could be identified and also the supernatant fragments of GPIb and GPV. GPIIIa was also cleaved within a loop structure formed by disulfide bond(s). The fact that remnants of both GPIIb and IIIa are left on chymotrypsin-treated platelets which aggregate spontaneously with fibrinogen may indicate that a complex formed by these remnants constitutes the fibrinogen-binding site on platelets.

Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin.

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.

Monoclonal antibodies directed against human α-thrombin and the thrombin-antithrombin III complex

Human α-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against α-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG 2a, and one was IgG 1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.

Effect of the Binding of Anti‐Zwa Antibodies on Platelet Function

Anti-Zwa antibodies can induce a Glanzmann-like platelet dysfunction in normal Zwa-positive platelets. Although a normal shape change and release reaction was always recorded in response to adenosine diphosphate and collagen, the aggregation on these inducers could be completely inhibited by anti-Zwa antibodies. Analogous to the platelets from patients with Glanzmann's thrombasthenia, normal platelets sensitized with anti-Zwa did not associate with fibrinogen upon exposure to adenosine diphosphate. Our data indicate that the binding site for fibrinogen is closely associated with the Zwa-antigenic determinants on the platelet membrane glycoproteins and thus with glycoprotein IIIa which is known to carry Zwa.

Retention of the glycoprotein IIb-IIIa complex in the isolated platelet cytoskeleton. Effects of separable assembly of platelet pseudopodal and contractile cytoskeletons.

To investigate the association of the putative platelet fibrinogen receptor (glycoprotein IIb-III(a) with the cytoskeleton, 125I-surface labeled human platelets washed by gel-filtration were activated under conditions which allow selective assembly of the platelet cytoskeleton. The four conditions were activation with arachidonate or phorbol 12-myristate 13-acetate (PMA) with and without pretreatment with cytochalasin E. Activation with arachidonate generates a complete cytoskeletal core (pseudopodal and contractile elements) while PMA activation forms only an actin plus actin-binding protein pseudopodal core. Pretreatment with cytochalasin E leads to actomyosin contractile core formation if arachidonate activated, and essentially blocks cytoskeletal development if PMA activated. Cytoskeletal cores from arachidonate or PMA-activated platelets retained 26 (+/- 3%) of the total 125I-IIIa. Pretreatment with cytochalasin E followed by arachidonate or PMA activation reduced the 125I-IIIa retention to near control levels (unactivated platelets: 4 +/- 2%). The role of aggregation vs. receptor occupancy in the retention of IIb-IIIa was assessed by activation of platelets with arachidonate in the presence of fibrinogen fragment D (0.6-12 mg/ml). Aggregation was blocked by increasing concentrations of fragment D reagent while cytoskeletal assembly was not altered. The IIIa retention correlated with extent of aggregation with maximal retention corresponding to full aggregation. To determine if cytoskeletal development is necessary for the expression of the fibrinogen binding site, binding studies were performed with unlabeled platelets and 125I-fibrinogen. The mean number of binding sites and the mean dissociation constant were not significantly different among the four activation conditions. Although the development of a platelet cytoskeletal core is not required for the expression of the fibrinogen binding site, the retention of the glycoprotein IIb-IIIa complex is dependent on fibrinogen-supported aggregation as well as the formation of the pseudopodal cytoskeleton.

Platelet aggregation: its relation with ADP-induced fibrinogen binding to platelets and ADP-related membrane enzyme activities.

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.

Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain.

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.

Bovine alpha- and beta-thrombin. Reduced fibrinogen-clotting activity of beta-thrombin is not a consequence of reduced affinity for fibrinogen.

beta-Thrombin, a product of the limited proteolysis of alpha-thrombin, is characterized by greatly reduced fibrinogen-clotting activity as compared to alpha-thrombin but with unchanged activity toward ester substrates. The present study was designed to elucidate the basis for the changes in the catalytic activity resulting from the conversion of bovine alpha-thrombin to bovine beta-thrombin. Fibrinogen was utilized as a competitive inhibitor in the hydrolysis of a peptide nitroanilide substrate by bovine alpha- and beta-thrombin. The Ki values obtained for fibrinogen in these experiments were similar for alpha- and beta-thrombin (about 10 microM). Similar values for Ki were obtained when fibrinogen was used to inhibit the inactivation of bovine alpha- and beta-thrombin by diisopropylphosphorofluoridate. These experiments suggested that the conversion of bovine alpha- to beta-thrombin does not affect the fibrinogen-binding site on thrombin. Differences in the reactivity of functional groups at the active site were then explored. beta-Thrombin was observed to undergo modification at the active site histidine at a slower rate than that of alpha-thrombin when reacted with either tosyllysyl chloromethyl ketone or diethyl pyrocarbonate. It is suggested that the difference in the fibrinogen-clotting activity of these two forms of thrombin can result from changes in the reactivity of the active site histidine residue.

Common protective antigens of group A streptococcal M proteins masked by fibrinogen.

The influence of fibrinogen on the opsonization of Group A streptococci by type-specific and cross-reactive anti-M protein antisera was investigated. As previously reported for type 24 streptococci, fibrinogen inhibited the complement-mediated opsonization of types 5, 6, and 19 organisms. Rabbit antisera against large peptide fragments of purified homologous M proteins (pep M proteins) overcame the anti-opsonic effect of fibrinogen in a dose-dependent manner. In the presence of optimal amounts of antibody, bacterial uptake by PMN was equal in serum and plasma, and greater than could be obtained in serum in the absence of antibody. Polyclonal anti-pep M sera contained antibodies directed against fibrinogen-binding as well as fibrinogen-nonbinding sites or regions of the M protein molecule. Three cross-reactive anti-pep M sera included antibodies directed against fibrinogen binding sites or regions of the cross-reacting M proteins. In the two sera studied in detail, these antibodies accounted for a large part of the cross-reacting anti-M antibody present in the sera. We suggest that fibrinogen binding sites on different serotypes of M protein may be structurally and therefore antigenically similar. Conservation of fibrinogen binding sites on M proteins may be related to their protective anti-opsonic function.

Characteristics of an ADP receptor mediating platelet activation.

Adenosine diphosphate (ADP) is known to induce platelet shape change, aggregation and fibrinogen binding, followed by secretion. These processes are mediated by the binding of ADP to an externally oriented protein of the platelet plasma membrane. An affinity analog of ATP, a competitive inhibitor of the action of ADP, has been utilized to probe the structure and function of this receptor. FSBA (5'-p-fluorosulfonylbenzoyl adenosine) covalently modifies a single protein in intact platelets with Mr = 100 000 and concomitantly inhibits platelet shape change, aggregation and fibrinogen binding. Studies on platelet membranes demonstrate non-covalent association of ADP-binding protein with actin which is also labeled by FSBA but only in isolated membranes. This finding suggests a structural and functional coupling of the receptor to the contractile process. The putative ADP receptor covalently modified with FSBA is cleaved by chymotrypsin, a process that reverses the inability of the platelets to bind fibrinogen. Thus, the Mr = 100 000 polypeptide may be involved in the proteolytic exposure of fibrinogen binding sites on the platelet surface. The ability of FSBA to inhibit platelet aggregation and fibrinogen binding by prostaglandin H2 derivatives and epinephrine suggest that ADP is involved in these processes. However, the interaction is not at the receptor level since shape change, stimulated by PGH2 derivatives and yohimbine (epinephrine antagonist) binding are unaffected by FSBA. Finally, the action of ADP to inhibit PGE1- or PGI2-stimulated adenylate cyclase appears to be mediated by a receptor distinct for the protein modified by FSBA.

Characterization of the Thrombin-Induced Contraction of Vascular Smooth Muscle

Purified human alpha-thrombin induced a sustained contraction of isolated rabbit aorta and dog coronary arteries. These vascular tissues also exhibited a refractoriness towards a second thrombin exposure. The extent of tachyphylaxis exhibited by the aorta correlated with the initial concentration of thrombin and the length of time the tissue was exposed to thrombin. The thrombin-induced contraction in the aorta was not blocked by phospholipase or cyclooxygenase inhibitors, but it was inhibited in the presence of hirudin, heparin, nitroglycerin, and nitroprusside. Nitroglycerin, nitroprusside, and hirudin also inhibited the contraction in the dog coronary artery. Ca++ channel blockers did not inhibit the thrombin-induced contraction in the coronary artery, although a small inhibition was observed in Ca++-free media. In both tissues, equivalent contractile responses were obtained using equimolar quantities of beta-, tetranitromethane-, and alpha-thrombin, even though the latter's coagulant activity was 30-40 times that of the modified thrombins. However, if the catalytic activity of thrombin was inhibited by modification with Tos-Lys-CH2Cl, hirudin, or heparin/antithrombin III, the vasoconstrictor activity was also lost. These studies suggest that alterations of the fibrinogen-binding site do not affect the contractile activity of thrombin. The contraction may be the result of a proteolytic interaction of the active site of the enzyme with vascular smooth muscle.

Enzymic activity of thrombin with partially reduced disulfide bonds

Partial reduction of thrombin disulfide bridges with dithiothreitol in the absence of denaturants inhibits the fibrinogen clotting but not the esterase activity of the enzyme. The clotting activity reappears on spontaneous air reoxidation of thrombin. As a result of the reaction with dithiothreitol, two disulfide bonds are cleaved in thrombin molecule inducing a small decrease of β-sheets in the secondary structure of thrombin. It may be concluded that this modification does not affect the catalytic site of thrombin but has influence upon the fibrinogen binding (recognition) site.

Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

Exposure of Platelet Fibrinogen-Binding Sites by Collagen, Arachidonic Acid, and ADP: Inhibition by a Monoclonal Antibody to the Glycoprotein Ilb-IIIa Complex

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I-fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029-0.045 microM) binding 1000-1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2-2.0 microM) binding 46,000-76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb-IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

Platelet aggregation in laminar flow. II. Shear rate and ADP-dependent effects of acetylsalicylic acid and indomethacin

The relative contributions of physiologic activators and shear rates (G) on the formation and stability of platelet aggregates arising from platelets treated with widely clinically used nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA) need to be characterized for conditions of low G believed to be important in vivo. In Part I of these studies it was found that platelets in human citrated platelet-rich plasma (PRP) undergo ADP-induced aggregation in laminar flow by apparently two distinct processes: a low ADP-dependent process, generating large visible aggregates stable only at low shear rates, and a higher ADP-threshold-dependent process yielding more complete and shear-resistant large aggregate formation. Similar studies of both macroaggregation (TA) and actual % aggregation (%PA) were made with ADP injections into PRP in the cone-in-plate device as a function of G and pre-treatments with ASA (200 μM) and indomethacin (10 μM). These drugs reduced %PA in part and yielded only reversible macro-aggregation for studies conducted at the higher [ADP] (4–6 μM) and at high G (≳ 120–150 sec −1), with no significant effects at low G (≲ 30 sec −1). The results are discussed in terms of a platelet stickiness factor ‘s’, ascribed to changes in fibrinogen binding sites reported for these drug actions, and related to in vivo flow and variable platelet activation conditions.

Further studies on the interaction between thrombin and GP Ib using crossed immunoelectrophoresis. Effect of thrombin inhibitors

The platelet surface protein GP Ib (glycocalicin-related protein) has been shown to be retarded by thrombin-Sepharose 4B in a crossed immunoelectrophoresis system. The interaction between GP Ib and thrombin was abolished when thrombin was blocked either at the active serine site with tosyl-lysine-chloromethyl-ketone (TLCK) or phenylmethylsulfonylfluoride (PMSF) or at the fibrinogen binding site (macromolecular binding site) with N-bromosuccinimide (NBS) or heparin, indicating that both sites have to be freely accessible for the retention of the glycocalicin-related protein by thrombin.

Demonstration of variable forms of the platelet factor 4 immunoprecipitate using crossed immunoelectrophoresis

Proteins with different electrophoretic properties were precipitated by a monospecific antiserum to platelet factor 4 either as a “line” or as a “peak” precipitate. The “line” form seen on crossed immunoelectrophoresis of whole platelets was retained when immobilized thrombin was included in the intermediate gel. The retention was partially abolished when thrombin had been blocked at the active serine site or at the fibrinogen binding site. The “peak” form seen on analysis of material secreted from platelets passed unaffected through thrombin-Sepharose. It is suggested that platelet factor 4 exixts in the platelets in a state different from that observed extracellularly after platelet secretion.

Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen.

We have recently reported the isolation of purified platelet membrane glycoproteins IIb and IIIa and the generation of monospecific antisera to these membrane proteins. Using these monospecific antisera in an enzyme-linked immunosorbent assay system, it is no demonstrated that glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a complex with purified human fibrinogen. The formation of this GPIIb-GPIIIa fibrinogen complex is calcium dependent, fibrinogen specific, saturable, and inhibited by specific amino sugars and amino acids. These observations suggest that the GPIIb-GPIIIa macromolecular complex on the platelet surface acts under the proper physiologic circumstances as the fibrinogen binding site required for normal platelet aggregation.