Proteoglycan obtained from bovine aorta suppress thrombin-induced platelet aggregation.

Proteoglycan (PG), isolated and purified from bovine aorta (intima-media), consisted of 68.6% chondroitin 4/6-sulfate (CS 4/6-S), 30% dermatan sulfate (DS), 1.4% heparan sulfate (HS), and a trace of hyaluronic acid (HA). PG did not affect platelet aggregation induced by ADP, collagen, and epinephrine, but inhibited that induced by thrombin. Of the standard GAGs investigated, hyaluronic acid (HA) and CS-4/6-S slightly inhibited only thrombin-induced platelet aggregation. However, PG and standard GAGs did not affect the thrombin induced aggregation of washed platelets. The effect of PG after papain digestion on thrombin-induced platelet aggregation was less potent than that before. It is suggested by the results of this study that PG in the aorta inactivates plasma thrombin, probably by inhibiting thrombin activators or potentiating substances which inactivate thrombin and that these effect of PG would be mainly due to PG-DS and partly due to PG-HS.     

The role of regioselectively sulfated and acetylated polysaccharide coatings of biomaterials for reducing platelet and plasma protein adhesion.

A brief survey is given about the role of natural polysaccharides such as heparin (HE), heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) in reducing blood coagulation and their potential use as athrombogenic coatings in the development of tailor-made athrombogenic biomaterials. Furthermore, known literature and new results about platelet adhesion on regioselectively modified polysaccharides such as HE, chitosan with HE-like functional groups, and sulfated cellulose are presented in two different perfusion systems at different shear rates. Regioselectively modified polysaccharides were tested as coatings of two polymers. The strongest influence on platelet adhesion was observed when the three regioselectively modified polysaccharides contained 6-O-sulfo- groups. No or little influence was seen with 3-O-sulfo- groups. A variable effect on platelet adhesion was found in position 2. N-sulfo- groups in HE induced a medium platelet adhesion, and O-sulfo- groups of iduronic acid moiety in HE induced none. Cellulose containing 2-O-sulfo- groups induced little platelet adhesion, and 2-N- sulfo- groups in chitosan induced a variable platelet adhesion response, depending on the N-SO(3)/NAc ratio. Preliminary plasma protein adhesion measurements on immobilized HE derivatives with four different fluorescence-labeled plasma proteins showed a regioselective influence with serum albumin and fibrinogen. 6-O-desulfated HE gave the strongest reduction in protein adsorption followed by N-desulfation and 2-O-desulfation; the lowest reduction was observed with 3-O-desulfation. Tailor-made athrombogenic coatings of HE should not carry high amounts of 6-O-sulfo- groups or of N-sulfo- groups. Regioselectively modified cellulose and chitosan may become suitable for tailor-made athrombogenic biomaterials when regioselective reactions are further optimized.     

Plasma and urinary levels of dermatan sulfate and heparan sulfate derived disaccharides after long-term enzyme replacement therapy (ERT) in MPS I: correlation with the timing of ERT and with total urinary excretion of glycosaminoglycans

Introduction

Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels.

Methods

Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5 years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test.

Results

The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state.

Conclusions

HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100 IU/kg/weekly.     

Anti-platelet effects of different phenolic compounds from Yucca schidigera Roezl. bark

Resveratrol (3,4',5-trihydroxystilbene) has been reported to have a variety of anti-inflammatory, anti-carcinogenic, anti-fungal and anti-platelet effects. It occurs naturally in different medicinal plants. Recently, resveratrol and other related phenolic compounds including trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene and yuccaols A and C were isolated from the bark of Yucca schidigera. The aim of the present study was to evaluate in vitro the effects of these compounds on platelet aggregation induced by thrombin and ADP. Pretreatment of platelets with resveratrol or other tested phenolics (1-25 microg/ml) slightly reduced platelet aggregation stimulated by 5 microM ADP (P < 0.05) or 10 microM ADP (P < 0.005). The comparison of the inhibitory effects of tested compound in thrombin-induced platelet aggregation revealed that phenolic showed even stronger antiplatelet actions than resveratrol. These compounds also had an inhibitory effect on the thrombin-induced enzymatic platelet lipid peroxidation determined as the level of thiobarbituric acid reactive substances.     

Biochemical and immunohistochemical analysis of glycosaminoglycans in inflamed and non-inflamed intestinal mucosa of patients with Crohn’s disease

OBJECTIVES: Changes in extracellular matrix glycosaminoglycans (GAGs) of the intestinal mucosa have been associated with inflammatory bowel disease. The aim of the present study was to follow the changes in GAGs metabolism during the progression from non-inflamed to inflamed intestinal colon of patients with Crohn's disease (CD), using direct biochemical analysis and specific immunohistochemistry against chondroitin/dermatan sulfate and heparan sulfate. DESIGN AND METHODS: The content of GAGs from inflamed and non-inflamed colon of eight patients with active CD was estimated by uronic acid per dry weight of tissue and analyzed by agarose gel electrophoresis and ion-exchange chromatography. Intestinal sections were stained using antibodies against dermatan sulfate/chondroitin 4-sulfate (DS/CS), heparan sulfate (HS), and ICAM-1 (CD54), and analyzed by confocal microscopy. RESULTS: There was a reduction in the amount of GAGs in the non-inflamed colon of patients with CD. In the inflamed colon, HS, CS and DS showed increased concentrations compared with the non-inflamed colon. GAGs showed a diffuse distribution in the lamina propria and in the basement membrane of both inflamed and non-inflamed mucosa of patients with CD. CONCLUSION: We observed a marked reduction in GAGs with altered patterns of distribution in the non-inflamed colon of patients with CD. The increase in the synthesis of GAGs observed in the inflamed colon may be a compensatory mechanism for the restoration of the integrity of the intestinal mucosa.     

Anti-platelet effects of different phenolic compounds from Yucca schidigera Roezl. Bark

Resveratrol (3,4',5-trihydroxystilbene) has been reported to have a variety of anti-inflammatory, anti-carcinogenic, anti-fungal and anti-platelet effects. It occurs naturally in different medicinal plants. Recently, resveratrol and other related phenolic compounds including trans -3,3',5,5'-tetrahydroxy-4'-methoxystilbene and yuccaols A and C were isolated from the bark of Yucca schidigera . The aim of the present study was to evaluate in vitro the effects of these compounds on platelet aggregation induced by thrombin and ADP. Pretreatment of platelets with resveratrol or other tested phenolics (1-25 w g/ml) slightly reduced platelet aggregation stimulated by 5 w M ADP ( P < 0.05) or 10 w M ADP ( P < 0.005). The comparison of the inhibitory effects of tested compound in thrombin-induced platelet aggregation revealed that phenolic showed even stronger antiplatelet actions than resveratrol. These compounds also had an inhibitory effect on the thrombin-induced enzymatic platelet lipid peroxidation determined as the level of thiobarbituric acid reactive substances.     

Human abdominal aortic aneurysm is closely associated with compositional and specific structural modifications at the glycosaminoglycan level.

Human abdominal aortic aneurysm (AAA) is a commonly occuring disease of blood vessels and is related to alterations in extracellular matrix molecules. In this study we report on the type and fine structural characterization of glycosaminoglycans (GAGs) present in AAA as compared with those present in normal abdominal aorta. Hyaluronan (HA), the galactosaminoglycans-chondroitin sulfate (CS) and dermatan sulfate (DS) with average molecular size (Mr) of 35-kDa-as well as heparan sulfate (HS) with Mr of 40-kDa were identified in both tissues. No significant intrabatch differences in total GAG content were identified in normal and aneurysmal aortas. Comparing, however, tissue composition and structure of GAGs between AAAs and normal aortas, significant differences (P < or = 0.001) were found. The overall GAG content in AAAs was approx. 60% lower than the normal ones. A 90% decrease in HS content, and 65 and 73% in CS and HA, respectively, were also recorded. In contrast, only a slight decrease in the amount of DS was noted (8%). Structural alterations in disaccharide composition of GAGs correspond mainly to significant decreases (P < or = 0.001) of HS-derived N-sulfated disaccharides, CS-derived 6-sulfated disaccharide and DS-derived disulfated disaccharides. These results demonstrate that the development of AAA is related to dramatic quantitative and structural modifications at the GAG level and this may well be attributed to the destruction of arterial wall architecture and further significant functional inadequacies of the tissue.     

Distribution and composition of glycosaminoglycans in the left human coronary arterial branches under myocardial bridge

The distribution and composition of glycosaminoglycans (GAGs) are reported in the anterior interventricular branch under the intermyocardial bridge (MB) and the ventricular branch without bridge, both from the left human coronary artery. Chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) were purified and quantified by a combination of electrophoretic migration and enzymatic degradation. The absolute amounts of GAGs in the intermyocardial bridge segment (MB) increased by 47%, when compared to the pre (PreMB) and post (PostMB) segments and the ventricular arterial branch (VB). Furthermore, the relative distribution of GAGs in the intermyocardial bridge segment differs when compared to the pre and post segments as well as in the ventricular arterial branch, due to a change in the proportion of DS and CS of 41.9 and 32.4%, compared to 36.4 and 39.7%, respectively. These findings give support to the possible involvement of GAGs in the intermyocardial bridge segment, avoiding local thrombus deposition, reducing atherosclerotic development and moreover giving protection against vessel deformation caused by the systolic pressure.     

Thrombin-, Epinephrine- and Collagen-Induced Platelet Aggregation Inhibited by α1-Acid Glycoprotein

α₁-acid glycoprotein (α₁-acid GP) isolated from human plasma was found to inhibit thrombin-induced aggregation of washed human platelets (final thrombin concentration 0.05 NIH U/ml), and inhibition was complete with physiological concentrations of the glycoprotein (1.0–1.5 g/l final conc.). The inhibitory effect seemed to occur immediately on thrombin addition, thus being similar to the effect of heparin previously observed. As opposed to heparin, however, α₁-acid GP did not affect spontaneous platelet aggregation. Furthermore, α₁-acid GP (in optimal concentrations) reduced the combined inhibitory effect of heparin and antithrombin III on thrombin-induced platelet aggregation, thus being consistent with the previous findings using heparin thrombin clotting time. Snyder & Coodley (1976) found α₁-acid GP to inhibit platelet aggregation induced by epinephrine and adenosine diphosphate in platelet-rich plasma. As we also found α₁-acid GP to inhibit collagen-induced platelet aggregation, this acid glycoprotein may possibly act as an inhibitor of the release reaction although fairly high concentrations (10 mg/ml final conc.) were needed for complete inhibition.     

Modulation of platelet and leucocyte function by a Chinese herbal formulation as compared with conventional antiplatelet agents

Platelet and leucocyte activity are important in the acute development of thrombosis and in the pathogenesis of ischaemic vascular disease. Dan Shen Di Wan (DS, Cardiotonic Pill or Dantonic(R) Pill) is one of the most commonly used Chinese herbal formulations for treating patients with atherosclerotic disease in China and several Asian countries. We studied the effect of DS on platelet and leucocyte function and compared the effects with conventional antiplatelet agents, cangrelor (ADP P2Y(12) receptor antagonist) and aspirin (acetyl salicylic acid, ASA). Measurements were made by platelet aggregation (%) and activation (CD62P %), platelet-monocyte conjugate formation (P/M, CD42a median fluorescence, mf), platelet-neutrophil conjugate formation (P/N, mf), and leucocyte activation (CD11b median fluorescence on monocytes and neutrophils, mf) in response to 3.3 micromol/L adenosine diphosphate (ADP), 1.0 micromol/L platelet activating factor (PAF), 5.0 micromol/L adrenaline and 0.5 microg/mL collagen. We also evaluated the effect of its main component, water soluble extract of salvia miltiorrhiza (SME) on intracellular calcium mobilization in platelets triggered by 10 micromol/L ADP, 10 micromol/L PAF, 2 microg/mL collagen and 15 micromol/L thrombin receptor activating peptide (TRAP). Overall DS showed inhibition of platelet aggregation, platelet activation, platelet-leucocyte conjugate formation and leucocyte activation in response to all the agonists apart from adrenaline (all p < 0.01). DS showed inhibition of platelet aggregation and leucocyte activation equivalent to cangrelor 100 nmol/L and ASA 100 micromol/L. SME dose-dependently inhibited intracellular calcium mobilization in platelets following stimulation with all the platelet agonists with maximum effective at 0.36 mg/mL (all p < 0.01). When used at 0.18 mg/mL its inhibitory effect was equivalent to cangrelor and ASA. We conclude that DS is a potential inhibitor of both platelet and leucocyte activation.     

Vitronectin inhibits blood platelet aggregation

The effect of vitronectin on platelet aggregation has been investigated. Vitronectin inhibited both thrombin- and ADP-induced platelet aggregation in a dose-dependent manner. A monoclonal antibody (MoAb) to vitronectin increased thrombin-induced platelet aggregation. This effect of the MoAb was not mediated via the platelet Fc-receptor, suggesting that the antibody directly counteracted the inhibitory effect of vitronectin on platelet aggregation. Like some other adhesive proteins such as fibrinogen, fibronectin, and von Willebrand factor, vitronectin contains the amino-acid sequence Arg-Gly-Asp (RGD) which enables binding to the platelet membrane glycoprotein complex IIb/IIIa (GPIIb/IIIa). The results of this study indicate that vitronectin can modulate the function of fibrinogen on platelet aggregation by interfering with the binding of fibrinogen to GPIIb/IIIa in activated platelets.     

Inhibition of Thrombin-Induced Platelet Aggregation by Crude and Highly Purified α1-Acid Glycoprotein

The inhibitory effect of crude and highly purified α₁-acid glycoprotein on thrombin-induced platelet aggregation were found to be identical.     

Thrombin interaction with human platelets. Potentiation of thrombin-induced aggregation and release by inactivated thrombin

The possibility that thrombin acts on platelets by a mechanism other than proteolysis was investigated. The proteolytic site of thrombin was modified with phenylmethylsulfonyl fluoride (PMSF). This modified enzyme did not induce platelet aggregation or the platelet release reaction. Platelets were then incubated with the inactivated enzyme (PMS-thrombin) and later with active thrombin. In this sequence of incubation, PMS-thrombin enhanced not only platelet aggregation induced by active thrombin but also the thrombin-induced release reaction. Preincubation with PMS-thrombin was essential for this enhancement as the inhibited enzyme did not affect aggregation if added after active thrombin. The effect of PMS-thrombin was limited to thrombin-induced reactions of the platelet. The inhibited enzyme had no effect on aggregation induced by adenosine diphosphate or collagen, or on thrombin-induced coagulation of fibrinogen. These results suggest (1) that both proteolytic and binding sites for thrombin are present on the human platelet plasma membrane; and (2) that interaction of thrombin with the binding site potentiates the activity of the proteolytic site.     

Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C.

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.     

Inhibition of coagulation and thrombin-induced platelet activities by a synthetic dodecapeptide modeled on the carboxy-terminus of hirudin

A synthetic, tyrosine-sulfated, dodecapeptide (BG8865) modeled on residues 53-64 of hirudin was found to elevate the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of human plasma in a dose-dependent manner. The most sensitive assay was the TT, which was prolonged 2 and 3 times control values at 2.2 and 4.1 micrograms/mL hirudin peptide, respectively. The sulfated dodecapeptide exhibited no dependency on antithrombin III as monitored by the APTT in the presence of sheep anti-human antithrombin III antibodies, and its activity was not neutralized by platelet releasates or platelet factor 4. In studies of thrombin-induced platelet activation, the hirudin peptide was found to block aggregation, serotonin release and thromboxane A2 generation. At thrombin concentrations of 0.25 U/mL, the IC50 (concentration resulting in 50% inhibition) for inhibition of platelet aggregation was 0.72 micrograms/mL peptide. Inhibition of TXA2 generation and serotonin release correlated closely with inhibition of aggregation. Using platelets from patients with clinically documented heparin-induced thrombocytopenia anticoagulant doses of heparin were found to induce platelet aggregation and thromboxane A2 generation. In sharp contrast, anticoagulant-equivalent doses of hirudin peptide had no effect on patient platelets, as evidenced by a lack of platelet aggregation and thromboxane A2 generation. These data provide compelling in vitro evidence that the hirudin peptide has several potential advantages over heparin, namely effective inhibition of thrombin-induced platelet activities, co-factor independence, insensitivity to endogenous heparin-neutralizing factors, and an apparent lack of direct or immune-mediated platelet stimulating properties.     

Antithrombotic effect of ticlopidine on He-Ne laser-induced thrombus formation in rat mesenteric microvessels.

The inhibitory effects of ticlopidine and acetylsalicylic acid (ASA) on thrombus formation in rat mesenteric microvessels were studied. The results were compared with the effect of the drugs on platelet aggregation in citrated whole blood. He-Ne laser-induced thrombus formation in arterioles and in venules was significantly inhibited by 100 mg/kg ticlopidine. In contrast, a higher dose of ASA (300 mg/kg) was needed to inhibit thrombus formation and the effects of ASA were observed only in arterioles and not in venules. In addition, although the inhibition by ASA in arterioles was not strong it followed a dose-dependent manner, suggesting that the differential effect of ASA on platelets and endothelium may not be evident in vivo. Ticlopidine and ASA strongly inhibited ADP-induced whole blood platelet aggregation, but not collagen- or thrombin-induced platelet aggregation.     

Inhibition of Human Platelet Aggregation by Plasmin Digests of Human and Bovine Fibrinogen Preparations: Role of Contaminating Factor VIII-related Material

Preparations of human fibrinogen, digested by plasmin, inhibited ADP-inducedplatelet aggregation; the inhibitory activity was confined to the small dialyzablefragments accumulating during the degradation. Purified large molecular weightfragments D and E had no effect on ADP-induced aggregation, but fragment E inhibited thrombin-induced aggregation.Extensively degraded bovine fibrinogenpreparations also inhibited platelet aggregation by ADP. Both human and bovinefibrinogen preparations were contaminated with factor VIII-related material(factor VIII-related antigen and factorVIII procoagulant activity, respectively);separation of factor VIII-related materialfrom human or bovine fibrinogen by gelchromatography and subsequent plasmindigestion of the fractions revealed thatthe inhibitory activity was mainly linkedto digested factor VIII-related material.This inhibitory activity was dialyzable.The effect of fibrinogen digests on plateletaggregation should therefore be reconsidered.

Submitted on May 4, 1973 Revised on January 17, 1974 Accepted on January 23, 1974     

Inhibitory effect of lysophosphatidylcholine and phospholipase A2-treated low density lipoproteins on receptor-dependent regulation of {Ca2+}i in platelets

Treatment of LDL with phospholipase A from bee venom resulted in formation of lipid-protein particles (pl2 LDL) with increased content of lysophosphatidylcholine (LPC). At the same time, composition of other lipids and protein structure were unaffected. Both pl-LDL and LPC abolished PAF-, ADP- and thrombin-induced Ca2+ elevation in platelets and platelet aggregation, while LDL had no effect on hormone-stimulated increase in the intracellular Ca2+ content ({Ca2+}i). The effect persisted in Ca2+-free medium, indicating that pl-LDL and LPC also abolish Ca2+ mobilisation from intracellular stores. Neither LPC nor pl-LDL changed platelet Ca2+ levels and inhibited platelet aggregation induced by thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ ATPase. The inhibitory effect depended on LPC concentration, incubation time and the structure of LPC: lysophosphatidylethanolamine and phosphatidylcholine produced no inhibitory effect. The half-maximum-effective concentrations were the same for LPC and pl-LDL (2-4 microM). The results obtained indicate that LPC and pl-LDL inhibit the receptor-dependent increase in Ca2+. It can be suggested that the effect of LPC is mediated by redistribution of the plasma membrane integral proteins, which leads to disintegration of the intracellular signalling systems.     

Functional abnormalities of heparan sulfate in mucopolysaccharidosis-I are associated with defective biologic activity of FGF-2 on human multipotent progenitor cells

In mucopolysaccharidosis-I (MPS-I), alpha-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2-FGFR1-HS interactions, resulting in defective FGF-2-induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS-cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.     

Antibodies against platelet membrane glycoproteins

Glycocalicin has been proposed as the common platelet receptor for both ristocetin-human VIIIR:WF and thrombin-induced platelet aggregation. Platelets which have lost glycocalicin do not respond to either ristocetin-human VIIIR:WF or bovine VIIIR:WF. Using antibodies to the platelet membrane glycoproteins Ia and Ib, IIb and IIIa, and glycocalicin we show that the Fab' fragments of anti-glycoproteins Ia and Ib and anti-glycocalicin IgG totally inhibited bovine VIIIR:WF-induced platelet aggregation, while those from anti-glycoproteins IIb and IIIa IgG were without effect. Thrombin-induced platelet aggregation was strongly inhibited by the Fab' fragments of anti-glycoproteins Ia and Ib IgG and anti-glycocalicin IgG, indicating that these glycoproteins play a major role in thrombin-platelet interaction. Fab' fragments of anti-glycoproteins IIb and IIIa IgG inhibited thrombin-induced platelet aggregation to a lesser extent implying that these glycoproteins are also somehow involved in the platelet response to thrombin perhaps as fibrinogen receptors. The data presented here give further support to the proposal that ristocetin-human VIIIR:WF and bovine VIIIR:WF share a common receptor on the platelet surface and indicate that this structure plays an important role in thrombin-induced platelet responses.