Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation.

Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.     

Deficiency of platelet membrane glycoprotein Ia associated with a decreased platelet adhesion to subendothelium: a defect in platelet spreading

A bleeding disorder with absent collagen-induced platelet aggregation and adhesion has been described in a patient whose platelets failed to express surface glycoprotein Ia. We studied the interaction of her platelets with subendothelium in an annular perfusion chamber and the interaction with purified human collagen type III in a rectangular perfusion system under flow conditions. Platelet adherence was almost completely absent both at low and high shear rates. The few platelets which adhered remained in the contact stage without subsequent spreading and aggregate formation. Addition of a monoclonal antibody, which was directed against the von Willebrand moiety of FVIII-VWF, to the blood, completely abolished platelet adherence at high shear rates and had a partial effect at low shear rates. These data indicate that von Willebrand factor plays a role in the initial attachment (contact stage) of platelets to subendothelium. We conclude that the bleeding disorder and excessively prolonged bleeding time in our patient are caused by a new specific defect of the platelet-vessel wall interaction.     

Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex

The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane.     

Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3

A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.     

Platelet membrane fluidity and aggregation of rabbit platelets

Aggregation of rabbit platelets from citrated plasma in response to ADP was directly correlated with platelet plasma membrane fluidity as determined by fluorescence depolarization measurements with the probe diphenylhexatriene. Rabbits were maintained for periods of 200 and 400 days on potentially hyperlipidemic diets (20% fat by weight) with varying levels of saturated and polyunsaturated fatty acids. Dietary variations were effective in modulating the mole percentage distribution patterns of the platelet phospholipid fatty acids. The major chemical control of membrane fluidity was the actual mass of unsaturated lipid in the cells and not simply the relative percentage distributions of such unsaturated fatty acids. Substantially higher phospholipid/protein ratios were observed upon analysis of platelets and platelet membranes from rabbits after 200- than after 400-day diet periods. Accordingly lipid structures were significantly more fluid in either whole platelets or membrane isolates at the end of the shorter diet period. The observations pertaining to the extent of aggregation and membrane fluidity are in consonance with the general role of membrane fluidity in controlling biological activity and support the concept that platelet aggregation is a membrane-associated phenomenon.     

A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia

We found a novel platelet aggregating factor in a patient with steroid- responsive immune thrombocytopenic purpura that is associated with defective collagen-induced platelet functions. The aggregating factor and platelet functions were analyzed. The patient, a 58-year-old female, had purpura and prolonged bleeding time despite adequate platelet counts (greater than 140,000/microL) after steroid therapy. The patient's platelets responded normally to all agonists except collagen. Platelet adhesion to collagen fibrils was decreased. The patient's plasma induced irreversible aggregation and ATP release in normal platelet-rich plasma (PRP). This platelet aggregating factor was found in F(ab')2 fragments of the patient's IgG, which caused thromboxane B2 synthesis, elevation of cytoplasmic Ca2+ levels, and phosphorylation of 40 kDa protein in normal platelets. Platelet aggregation by the patient's IgG was inhibited by prostacyclin, dibutyryl cAMP, diltiazem, disodium ethylenediaminetetraacetate, and antimycin A plus iodoacetate, but ADP scavengers, cyclo-oxygenase inhibitors, and heparin had little or no effect. The aggregating activity of the patient's IgG absorbed to and eluted from normal platelets. The patient's Fab fragments did not induce platelet aggregation in eight of ten normal PRP but specifically inhibited aggregation induced by collagen and by the patient's IgG. The major component of an immunoprecipitate made with the patient's IgG from radiolabeled membrane proteins of normal platelet extract had a 62 kDa mol wt, while no such precipitate appeared in extracts of the patient's platelets. These results indicated that platelet aggregation by the patient's IgG was induced by the reaction of an antibody with a specific antigen on the normal platelet membrane through stimulus- response coupling. This antigen may be a collagen receptor on the platelet, most likely a polypeptide of 62 kDa under reducing condition. The defect of collagen-induced aggregation of the patient's platelets seemed to be due to alteration of the membrane protein related to this putative collagen receptor.     

Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation.

We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.     

Evidence for a storage pool defect in platelets from cirrhotic patients with defective aggregation.

The mechanisms underlying the defective platelet function in cirrhotic patients were investigated. Eleven cirrhotic patients with mild disease (group 1), 20 patients with severe cirrhosis (group 2), and 31 controls were studied. Platelet aggregation was significantly reduced in cirrhotics compared with controls. Compared with controls, cirrhotic patients in group 2 showed a significant reduction in the total content of adenosine triphosphate (57.8 +/- 7.8 vs. 26.1 +/- 6.3 mumol/10(11) platelets; P less than 0.05), 5-hydroxytryptamine (285 +/- 26 vs. 104 +/- 38 nmol/10(11) platelets; P less than 0.05), beta-thromboglobulin (2129 +/- 120 vs. 1223 +/- 161 ng/10(8) platelets; P less than 0.01), and platelet factor 4 (1389 +/- 108 vs. 805 +/- 176 ng/10(8) platelets; P less than 0.05). In patients with severe disease, an increase in plasma beta-thromboglobulin-platelet factor 4 ratio, an index of in vivo platelet activation, was observed (controls, 3.50 +/- 0.50; group 1, 4.02 +/- 0.80; and group 2, 6.59 +/- 1.15). Our data indicate the existence of a platelet storage pool defect, which may favor the bleeding tendency of cirrhotic patients.     

Red blood cells mediate spontaneous aggregation of platelets in whole blood

The influence of red blood cells on spontaneous platelet aggregation (SPA) has been studied ex vivo. Platelet aggregation was quantified by measuring the fall in single platelet count using a new whole blood platelet counter. When aliquots of whole blood and autologous platelet rich plasma (PRP) were roller-mixed at 37 degrees C, a marked fall in platelet count occurred in whole blood due to SPA but platelet count remained almost unchanged in PRP. When blood from healthy young controls, aged 20-35 years, was compared with healthy old controls, aged 48-80 years, and patients with thrombotic complications, the extent of SPA was in the order: thrombotic patients greater than old controls greater than young controls. Prostacyclin and the new stable prostacyclin analogue Iloprost, at 8 nM effectively inhibited SPA. 2-Chloroadenosine (10 microM) which is an inhibitor of ADP-induced platelet aggregation was also an effective inhibitor of SPA. Acetylsalicylic acid (56 microM) and the thromboxane A2 receptor blocker BM13.177 (0.5 microM) only partially inhibited SPA. ADP from red blood cells is suspected to mediate red cell-induced SPA. However, the possibility that the red cells have an important physical role in SPA cannot be ruled out.     

Efficient tyrosine phosphorylation of proteins after activation of platelets with thrombin depends on intact glycoprotein Ib

The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.     

Efficient tyrosine phosphorylation of proteins after activation of platelets with thrombin depends on intact glycoprotein Ib

The role of platelet glycoprotein Ib as a thrombin receptor has been often a subject of controversy. We have investigated the role of the thrombin receptors, GPIb and protease-activated receptor (PAR)-1. Tyrosine phosphorylation in whole platelet lysates and in cytoskeletal extracts was evaluated after activation with thrombin and with the thrombin receptor-activating peptide (TRAP). Different experimental approaches were applied including: (i) congenital deficiency of platelet GPIb (Bernard Soulier syndrome, BSS), (ii) antibody to GPIb (AP1), (iii) selective protease cleavage (metalloprotease), and (iv) antibody to (PAR)-1. After activation of control platelets with thrombin or TRAP, multiple proteins became tyrosine phosphorylated in platelet lysates and some of them associated with the cytoskeletal fraction. These effects were absent in BSS platelets. Presence of AP1 or metalloprotease treatment showed an inhibitory effect when platelets were activated with a low concentration of thrombin or TRAP. Blockade of PAR-1 with a specific antibody, SPAN 12, inhibited platelet response to both agonists. This study reinforces the hypothesis that GPIb is the high-affinity receptor for thrombin. The signaling mechanisms occurring through tyrosine phosphorylation of proteins triggered by thrombin seem to be dependent on intact GPIb. Moreover, our results indicate that both receptors, GPIb and PAR-1, are necessary to achieve a full platelet response to thrombin.     

Relationship between intracellular calcium-dependent process and protein-tyrosine phosphorylation in human platelets: studies of platelets from a patient with defective A23187-induced platelet aggregation

We had postulated that in a patient with defective calcium ionophore (A23187)-induced platelet aggregation, whose platelets showed normal intracellular Ca2+ mobilization in either the presence or absence of extracellular Ca2+ in response to A23187. A defect was present in an intracellular calcium-dependent process. We have now investigated whether the agonist-induced protein-tyrosine phosphorylation (PTP) was altered. Protein-tyrosine phosphorylation (PTP)-induced by A23187 in the patient's platelets was greatly diminished but that induced by thrombin was almost normal. These results suggest that an intracellular calcium-dependent process plays a fundamental role in A23187-induced PTP, whereas it does not in thrombin-induced PTP.     

Evidence for a role for Galphai1 in mediating weak agonist-induced platelet aggregation in human platelets: reduced Galphai1 expression and defective Gi signaling in the platelets of a patient with a chronic bleeding disorder

We have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.     

alpha-Actinin and membrane glycoprotein IIIa are different proteins in human blood platelets.

It has been suggested that a platelet protein that is very similar to muscle alpha-actinin is identical to the membrane glycoprotein IIIa (GPIIIa) of platelets and is responsible for anchoring actin filaments directly into the plasma membrane of platelets. To determine if alpha-actinin and GPIIIa are related in platelets, we analyzed the purified proteins on 5% sodium dodecyl sulfate/polyacrylamide gels. The two proteins differ in mobility in both the unreduced and reduced states, and they stain differently with silver stain. In addition, alpha-actinin is a prominent component of the detergent-insoluble cytoskeletons of platelets, whereas GPIIIa is absent from these structures. By using monospecific antisera to the individual proteins, it was demonstrated that alpha-actinin and GPIIIa are immunologically distinct. We conclude that alpha-actinin and GPIIIa are different proteins in human blood platelets and that it is unlikely that alpha-actinin is an integral membrane protein.     

Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane

Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.     

Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression.

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')2 fragments 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.     

Isolation and structural characterization of the polypeptide subunits of membrane glycoprotein IIb-IIIa from human platelets

We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N- linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.