Platelet Membrane Glycoprotein Ib Research Articles

The management strategy for von Willebrand disease

The von Willebrand factor (VWF), one of the largest plasma proteins in mammals, is an essential factor in primary hemostasis. It forms a bridge between platelet membrane glycoprotein Ib and extracellular matrix constituents, thereby playing a critical role in platelet adhesion to vascular injury sites. Its deficiency, known as von Willebrand disease is the most common inherited bleeding disorder. VWF is also a carrier protein for blood clotting factor VIII, an interaction required for normal factor VIII survival in circulation. As its symptoms are less severe than those of hemophilia and due to its wide variety, a large number of cases may not have actually been diagnosed. In the selection of therapeutic measures and patient management for bleeding symptoms, introduction of recombinant VWF concentrates is expected to expand the range of therapeutic options.

Inhibition of platelet aggregation and blood coagulation by a P-III class metalloproteinase purified from Naja atra venom

Snake venom metalloproteinases (SVMPs) are an important component in viperid and crotalid venoms, and these SVMPs play important and versatile roles in the pathogenesis of snakebite envenoming. The SVMPs from elapid venoms are not well elucidated compared with those from viperid and crotalid venoms. Atrase B is a nonhemorrhagic P-III SVMP purified from the Naja atra venom, which possesses a weak fibrinogenolytic activity. In this paper, the activity and mechanism of atrase B against platelet aggregation and blood coagulation were investigated. The in vitro assay showed that atrase B remarkably inhibited ristocetin- and thrombin-induced platelet aggregation by cleavage of the platelet membrane glycoprotein Ib, and the coagulation of normal human plasma, which may be caused by inhibiting coagulation factor VIII predominantly. When atrase B was intravenously injected into rats at doses of 0.05 and 0.30 mg/kg, the activated partial thromboplastin and the thrombin times were significantly prolonged in a dose-dependent manner. Similarly, the fibrinogen level decreased, but only a high dose of atrase B showed remarkable activity against platelet aggregation. Results suggested that anticoagulation was a more important function of atrase B compared with its activity against platelet aggregation. These results indicated that atrase B may play an important role in the anticoagulant properties of Naja atra venom. In addition, atrase B may be a potent anticoagulant agent because its effectiveness in vivo against platelet aggregation and blood coagulation.

Relative analysis of platelet activation with bleeding risk in patients with primary immune thrombocytopenia

目的研究原发免疫性血小板减少症（ITP）患者血小板活化状态和出血程度的相关性。方法采用ITP特异性出血评价工具（ITP-BAT）对43例ITP患者进行出血评分及出血程度分级，应用流式细胞术检测二磷酸腺苷（ADP）激活前后血小板膜糖蛋白（GP）Ⅰb、Ⅱb/Ⅲa和P选择素表达。分析GPⅠb、GPⅡb/Ⅲa、P选择素表达与血小板计数、未成熟血小板比例的相关性；分析GPⅠb、GPⅡb/Ⅲa、P选择素表达与ITP患者出血程度的相关性，以及对中度以上出血患者风险评估的应用价值。结果ITP患者激活前血小板GPⅡb/Ⅲa、P选择素表达均高于正常对照组（65.69±10.73对7.16±0.99，t=4.130，P<0.001；15.43±1.41对12.55±1.03，t=2.070，P=0.043），GPⅠb表达低于正常对照组（240.11±24.93对295.11±22.15，t=2.417，P=0.020）。ITP患者ADP激活后血小板GPⅡb/Ⅲa表达高于正常对照组（133.96±12.17对39.67±4.99，t=5.256，P<0.001），而P选择素、GPⅠb表达低于正常对照组（37.09±3.94对109.77±23.66，t=3.901，P<0.001；149.06±19.14对205.73±21.00，t=2.070，P=0.043）。ADP激活后GPⅠb表达、ADP激活前后P选择素表达及ADP激活前后GPⅠb、P选择素表达比值与血小板计数相关（P<0.05）。ADP激活后P选择素表达、ADP激活前后P选择素表达比值与未成熟血小板比例相关（P<0.05）。ITP患者ADP激活后GPⅠb表达、ADP激活前后P选择素表达及ADP激活前后P选择素、GPⅡb/Ⅲa、GPⅠb表达比值在不同出血程度之间差异有统计学意义（P<0.05）。ADP激活前后GPⅠb表达比值是ITP患者中度以上出血的正性影响因素（OR=3.05，P=0.011），ADP激活前后P选择素、GPⅡb/Ⅲa表达比值是ITP患者中度以上出血的负性影响因素（OR=0.32，P=0.023；OR=0.04，P=0.006）。结论血小板活化指标能较准确地评估ITP患者出血程度，可以作为治疗参考指标和疗效观察指标。

Balancing the Expression and Production of a Heterodimeric Protein: Recombinant Agkisacutacin as a Novel Antithrombotic Drug Candidate

Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and β-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and β-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris.

First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus

CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

IntroductionThe multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells’ surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. Material and MethodsVWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. ResultsTo investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. ConclusionsOur assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants.

The Co-Influence of VWD Type 2B and Type 2M Mutations and Platelet GPIbα On the Susceptibility of VWF to ADAMTS13 Under Shear Stress

Abstract 1108 Introduction:Von Willebrand Factor (VWF) is one kind of vital proteins involved in thrombosis, which exists in the form of multimeric glycoprotein in plasma of human. The size of VWF multimer is down regulated by metalloproteinase ADAMTS13. This physiological process is modulated by multiple factors. It was reported that the binding of platelets or GPIbα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to recombinant A1A2A3 segment in the condition of static state. Due to the mutations, VWF synthesized by patients of von Willebrand disease (VWD) type 2B and type 2M may enhance and decrease the binding of platelet membrane glycoprotein Ib (GPIb), respectively. This study aims to observe the influence of 2B/2M VWD mutations and recombinant GPIbα active fragment on the susceptibility of VWF to ADAMTS13, in the condition of high shear stress. Materials and Methods:We constructed and expressed different forms of recombinant full-length human VWF (rhVWF) including three type 2B mutations (P1337L, H1268D, R1308C), three type 2M mutations (M740I, R1205H, D1302G) and wide-type (WT) in Hela cells. Recombinant human ADAMTS13 (rhADAMTS13) and GPIbα (H1-V289) were both from our own lab. The different types of rhVWF were characterized by multimeric analysis and ristocetin-induced platelet aggregation (RIPA). We use rhADAMTS13 to digest seven full-length rhVWF in the reaction Buffer (20 mM Tris-HCl, 150mM NaCl, pH7.4, 1% bovine serum albumin) in the absence and presence of GPIbα (H1-V289). To develop a more physiological assay under fluid shear stress, we assessed the proteolytic cleavage of rhVWF by rhADAMTS13 under constant vortexing (2,500 rpm) in a PCR tube mini-mixer that mimics arterial fluid shear stress (30 dynes/cm2). The 176kDa fragment resulting from the cleavage of rhVWF at the Y1605-M1606 bond by rhADAMTS13 was visualized by 5% SDS-PAGE under reducing conditions and Western blot analysis. Results:SDS-agarose gel electrophoresis showed that all rhVWF mutants were in the similar multimeric distribution to the rhVWF-WT. In the RIPA assay, type 2B rhVWF exhibited clearly enhanced RIPA responsiveness with ristocetin at 0.5 mg/ml (R1308C > P1337L > H1268D > WT), while type2M rhVWF showed decreased RIPA responsiveness with ristocetin at 1.0 mg/ml (WT > M740I > R1205H > D1302G). At physiological salt concentration (150nM NaCl), type 2B mutants exhibited more susceptibility to rhADAMTS13 than WT and type 2M mutants in the absence of GPIbα (H1-V289). In the presence of GPIbα (H1-V289), the higher susceptibility of mutants to rhADAMTS13 was detected, which decreased in the following order: type 2B > WT > type 2M, whereas no obvious enhancement happened to the two type 2M mutants (R1205H and D1302G). Conclusions:Our study further confirmed that platelet GPIbα binding to VWF A1 domain could increase the susceptibility to ADAMTS13 at physiological conditions under high stress. The three type 2B mutations are all localized in the A1 domain and these mutations alone may also increase the susceptibility to ADAMTS13. While type 2M mutations M740I, R1205H and D1302G are localized in the D2, D3 and A1 domain, respectively. It is concluded that type 2M mutations localized close to or in the A1 domain may exhibit significantly lower susceptibility to ADAMTS13 in the presence of GPIbα (H1-V289). It may partially give the explanation that some VWD type 2M may display larger than normal VWF multimers in plasma. Disclosures:No relevant conflicts of interest to declare.

The impact of platelet membrane glycoprotein Ib alpha and Ia/IIa polymorphisms on the risk of thrombosis in the antiphospholipid syndrome

BackgroundPathogenesis of thrombus formation in antiphospholipid syndrome (APS) is not clear. Platelet membrane glycoprotein (GP) receptors play important roles in development of thrombosis. ObjectivesWe investigated the association between development of thrombosis in APS and polymorphisms of GPIb alpha variable number of tandem repeats (VNTR), Kozak, and GPIa C807T.Patients/MethodsSixty patients with APS (30 with proven thrombosis and 30 without thrombosis) and 63 controls were included. Presence of GPIa C807T polymorphism was determined with real-time PCR and GPIb alpha Kozak and VNTR polymorphisms by conventional PCR. ResultsFrequency of C807T TT genotype was significantly higher in APS with thrombosis than APS without thrombosis (p=0.023) and also in APS with multiple thrombi compared to APS without thrombi (p=0.023). Frequency of Kozak TC genotype was higher in APS with arterial thrombosis compared to APS with venous thrombosis, controls, and APS without thrombosis (p=0.03, p=0.0007, and p=0.0024 respectively). D allele frequency and D allele carrier state for VNTR were significantly less in APS than controls (p=0.0018 and p=0.0046 respectively). ConclusionsC807T TT genotype may confer a risk for thrombosis and Kozak TC genotype for arterial thrombosis. D allele of VNTR may protect from APS. No patients with C807T TT or Kozak TC genotypes carried the protective DD genotype of VNTR. These polymorphisms may increase risk for both arterial and venous thrombosis. The utility of prophylaxis with anti-platelet drugs in at least a subgroup of APS patients should be investigated with clinical trials.

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm 2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.

Unravelling the mechanism and significance of thrombin binding to platelet glycoprotein Ib

The main question concerning the mechanism of a-thrombin binding to platelet membrane glycoprotein (GP)Ib is whether it involves both thrombin exosite I and exosite II. The solution of two independent crystal structures suggests alternative explanations that may actually reflect different modes of binding with distinct pathophysiological significance. With respect to function, it is still unclear whether thrombin binding to GPIb promotes procoagulant and prothrombotic pathways of response to vascular injury or limits such responses by sequestering, at least temporarily, the active enzyme. We review here published information on these topics and touch upon ongoing studies aimed at finding definitive answers to outstanding questions relevant for a better understanding of thrombosis and haemostasis.

Variable number of tandem repeats polymorphism of platelet glycoprotein Ib α in Chinese people and CC genotype with aspirin sensitivity in patients with cerebral infarction

To study the prevalence of variable number of tandem repeats (VNTR) polymorphism in platelet membrane glycoprotein (GP) Ib alpha in a Chinese Han population and to determine the relationship between VNTR polymorphisms and aspirin resistance. Three hundred healthy individuals and 110 patients with cerebral infarction volunteered to participate in this study. The genotype status of all participants was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. Platelet aggregation in patients with cerebral infarction receiving aspirin (100 mg/day) for at least 7 days, was measured by optical transmission aggregometry. Only three alleles of GP Ib alpha, namely, B, C and D, were found. Type A was not found in the Chinese Han participants. Aspirin-sensitive patients were significantly more often of CC genotype than aspirin-semi-responders. Only three types of alleles B, C and D were detected in the north-eastern region of China. The CC genotype of the VTNR polymorphism in GPIb appears to be more sensitive to the inhibitory action of low-dose aspirin.

Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis

von Willebrand factor (VWF) performs its hemostatic functions through binding to various proteins. The A1 domain of VWF contains binding sites of not only physiologically important ligands, but also exogenous modulators that induce VWF-platelet aggregation. Sulfatides, 3-sulfated galactosyl ceramides, that are expressed on oligodendrocytes, renal tubular cells, certain tumor cells and platelets, have been suggested to interact with VWF under some pathological conditions. The binding of VWF to sulfatide requires the A1 domain, but its binding sites have not been precisely identified. Here, we report that alanine mutations at Arg1392, Arg1395, Arg1399 and Lys1423 led to decreased VWF–sulfatide binding. These sites have been reported to be the binding sites for platelet membrane glycoprotein (GP) Ib and/or snake venom botrocetin, and, interestingly, are identical to the monoclonal antibody (mAb) NMC4 epitope previously reported to inhibit the VWF-GPIb interaction. We observed that NMC4 also inhibited VWF interaction with sulfatides in a dose-dependent manner. Thus, we conclude that VWF binding sites of sulfatide overlap those of platelet GPIb and botrocetin.

The change of serum leptin and its relationship with platelet membrane glycoprotein Ib in patients with coronary heart disease

The aim of this paper was to investigate the change of serum leptin and its relationship with platelet membrane glycoprotein Ib (GP Ib) in patients with coronary heart disease (CHD). The enrolled included 50 patients with CHD (CHD group) and 30 patients without CHD (control group) who were diagnosed by coronary angiography. The positive percentage and the average fluorescence intensity of platelet membrane GP Ib were detected by full-blood flow cytometry. Serum leptin was detected by enzyme linked immunosorbent assay. The positive percentage and the average fluorescence intensity of platelet membrane GP Ib in the CHD group were significantly lower than those in the control group (P < 0.05). After correcting the differences of systolic blood pressure, body mass index (BMI), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting glucose, PPBS, fasting insulin and quantitative insulin sensitive index, serum leptin level in the CHD group was significantly higher than that in the control group (P < 0.05). Single factor correlative analysis revealed that serum leptin in CHD patients was negatively correlated with the average fluorescence intensity of platelet membrane GP Ib (P < 0.05). Multifactorial stepwise regression analysis showed that serum leptin in CHD patients was independently negatively correlated with the average fluorescence intensity of platelet membrane GP Ib (P < 0.05). Logistic analysis demonstrated that serum leptin was independently correlated with the risk of CHD (P < 0.05). Hyperleptinemia was verified in CHD patients. The increase of serum leptin could affect blood platelet activation. Hyperleptinemia may play an important role in the pathogenesis of CHD.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin α and β subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC 50 value of 0.35 μM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.

Redistribution of Activation-Dependent Antigens on the Platelet Surface In Vivo and In Vitro.

Platelets play a crucial role in thrombosis, platelet activation induces an alteration of platelet membrane glycoproteins between the platelet surface and surface connected canalicular system (SCCS). In an attempt to better understand this process and assess its significance in the study of thrombosis disease, we used flowcytometry for detecting the redistribution of antigens on the platelet surface following activation. The expression of platelet membrane glycoproteins (GP) Ib, IIIa and P-selectin as well as the platelet agglutination induced by ristocetin were measured in 20 normal subjects, whose platelets were stimulated by the peptide SFLLRNP(TRAP, thrombin receptor activated peptide) at different time points (0~30minute). The same antigens were also analyzed in whole blood in 30 patients with cerebral infarction. As expected, a stable increase of P-selectin was obtained from 2 minute upon TRAP activation, while a reversible reduction of GPIbα and agglutinability by ristocetin were showed at the same time, with a maximum decrease at 2 minute, then followed by a return of GP Ibα to platelet surface. In addition, the platelet membrane GPIIIa was not significantly changed in the initial stage after platelet stimulation and increased by degrees 10 minutes later. Compared with those in normal group, there was an apparent increase of P-selectin and a lower expression of GPIb in the patients with cerebral infarction, while GPIIIa was not significantly changed in all the samples. In summary, TRAP can mediate platelet activation, which leads to the redistribution of GPIb and GPIIIa in a time-dependent manner, together with the release of P-selectin. Meanwhile, the change of activation-dependent antigens on the platelet surface could be an important index reflecting the platelet activation in vivo.

Platelet glycoprotein I(b)alpha and integrin alpha2 beta1 polymorphisms: gene frequencies and linkage disequilibrium in a population diversity panel.

Genetic variants in the GP1BA and ITGA2 genes have been proposed as potential modifiers for arterial vascular disease and bleeding disorders. Since ancestry may play an important role in the prevalence of these variants, we sought to determine their allele frequency and linkage disequilibrium in a collection of 1064 DNA samples from 51 ethnic groups. We studied haplotypes of ITGA2 defined by single nucleotide substitutions at positions -52, 807, and 1648, and GP1BA variants defined by sequence changes in positions -5 (Kozak), 1018 (T145M, HPA-2) and 1285 (VNTR A, B, C and D). Frequency of haplotypes of ITGA2 showed considerable variation across the different groups, with a higher prevalence of the haplotype -52C or T/807C/1648A observed in African compared with caucasian and Asian populations. The haplotypes 52C/807T/1648A and -52T/807T/1648A were not observed in caucasians or South Americans. While relative frequencies of the GP1BA Kozak alleles were comparable across groups, the methionine allele (HPA-2b) showed a higher frequency in Africa (0.26) than in the other groups. We also observed a high prevalence of the VNTR B allele in the African and Israeli populations. Haplotype analysis revealed incomplete linkage disequilibrium between the HPA-2 and VNTR alleles. Incorporation of GP1BA variants into the set of SNPs already genotyped by the HapMap project disrupted the pre-existing haplotype block. These data provide a valuable resource for optimal selection of variants best tailored for association studies of vascular disease or bleeding disorders when examining individuals of different ancestral origins.

Combined administration of nitric oxide gas and iloprost during cardiopulmonary bypass reduces platelet dysfunction: A pilot clinical study

Thrombocytopenia and platelet dysfunction are major mechanisms of cardiopulmonary bypass-induced postoperative hemorrhage. This study evaluated the effects of low amounts of nitric oxide, iloprost (prostacyclin analog), and their combination administered directly into the oxygenator on platelet function, platelet-leukocyte interactions, and postoperative blood loss in patients undergoing coronary artery bypass grafting. Blood samples from 41 patients randomized to the control, nitric oxide (20 ppm), iloprost (2 ng x kg -1 x min -1 ), or nitric oxide plus iloprost groups were collected during cardiopulmonary bypass. Platelets and leukocytes were enumerated. Platelet membrane glycoprotein Ib and glycoprotein IIb/IIIa, P-selectin, platelet-derived microparticles, leukocyte CD11b/CD18 (Mac-1), and platelet-leukocyte aggregate were quantified by means of flow cytometry. Collagen and thrombin receptor-activating peptide-induced platelet aggregation in whole blood was analyzed by means of aggregometry. Both nitric oxide or iloprost attenuated cardiopulmonary bypass-induced thrombocytopenia, reduction of glycoprotein Ib and glycoprotein IIb levels, translocation of P-selectin, microparticle formation, Mac-1 upregulation, and suppression of collagen-induced aggregation. Nitric oxide plus iloprost was significantly more effective in preventing thrombocytopenia, microparticle formation, and P-selectin translocation. Moreover, this treatment preserved thrombin receptor-activating peptide-induced aggregation, which was not rescued by single treatments. Both nitric oxide and nitric oxide plus iloprost attenuated postoperative blood loss. Nitric oxide plus iloprost reduced the deleterious effects of cardiopulmonary bypass, such as thrombocytopenia, platelet activation, platelet-leukocyte aggregate formation, and suppression of platelet aggregative responses. The reduced postoperative bleeding observed with this treatment suggests that this is a new and clinically feasible therapeutic option for patients subjected to cardiopulmonary bypass.

A tetrameric glycoprotein Ib-binding protein, agglucetin, from Formosan pit viper: structure and interaction with human platelets.

Agglucetin, a tetrameric agglutination inducer from the Formosan pit viper, has been identified as a platelet membrane glycoprotein (GP) Ib agonist and directly agglutinated fixed-platelets in the absence of von Willebrand factor (vWf). Here, we resolved the complete cDNA sequences of agglucetin subunits (alpha(1), alpha(2), beta(1) and beta(2)) by molecular cloning. Each cloned cDNA encoding the leader peptide (23 residues) and the mature subunit (131/135/123/126 residues) shares a high degree of homology to each other and the C-type lectin-like GP Ib-binding proteins (BPs). Furthermore, agglucetin specifically caused platelet agglutination and surface exposure of integrin alpha(IIb)beta(3) with a GPIb-dependent manner in washed platelets, based on the observation that the enhanced expression of functional alpha(IIb)beta(3) was suppressed by a GPIb-cleaving metalloproteinase, crotalin. Pretreating platelets with staurosporine or BAPTA-AM also completely blocked the exposure of functional alpha(IIb)beta(3), suggesting that the activation of protein kinase C and intracellular calcium mobilization are involved in the GPIb-dependent signaling. In human platelet-rich plasma (PRP), agglucetin elicited sequential biphasic responses of platelet agglutination and aggregation in a GPIb- and alpha(IIb)beta(3)-dependent manner, respectively, implying that other cofactors may amplify platelet activation to trigger aggregation.

A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V.

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V -/- platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib--IX--V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y(12) ADP receptor (G(i)-coupled ADP receptor) is involved in this pathway, and that the P2Y(1) receptor (G(q)-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib--IX--V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib--IX via thrombin acting as a ligand, resulting in platelet activation.

A Novel Tetrameric Venom Protein, Agglucetin from Agkistrodon acutus , Acts as a Glycoprotein Ib Agonist

A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked alpha1, alpha2, beta1 and beta2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin. in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B, formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of API, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.