Convulxin Stimulation Research Articles

Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets.

Bruton's tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.

Platelet-Dependent Thrombin Generation Is Correlated with Phenotypic Severity in Severe Hemophilia a

Hemophilias A and B are classified into mild (>5-40%), moderate (2-5%) and severe (<1%) disease based upon plasma factor activity levels. Severity of bleeding is commensurate with the baseline factor levels in general. However, heterogeneity of bleeding pattern and severity in patients with severe disease is well described. The biologic mechanism responsible for this phenotypic heterogeneity has yet to be thoroughly explained. A role of platelets, as key contributors to hemostatic plug formation and coagulation, has been investigated in previous small cohorts of patients with severe hemophilia with variable results.A single-center cohort of 94 patients with severe hemophilia A were enrolled into an IRB approved study. Patients with concomitant bleeding disorders or thrombocytopenia (<100K/μL) were excluded. Flow cytometry was utilized to assess multiple aspects of platelet function, including 1) granule release (P-selectin) and integrin αIIbβ3 activation (PAC-1 high) at low and high doses of thrombin and the GPVI agonist convulxin and 2) procoagulant platelet formation (Annexin V high, PAC-1 low) with combined thrombin and convulxin stimulation. In 46 patients who had not received factor prophylaxis in the 96 hours prior to laboratory assessment, a thrombin generation profile, initiated by low concentration tissue factor, was obtained in both platelet-rich (PRP) and platelet-poor (PPP) plasma. To assess phenotypic severity, the Hemophilia Severity Score (Schulman S et al, 2008) was determined for each patient. The HSS score integrates bleeding rate, joint damage, and factor usage to obtain a composite measure of disease severity. Regression analysis was utilized to assess association (significance = p<0.05).No association with HSS was found with either granule release or integrin activation. This lack of association with bleeding phenotype in severe hemophilia is consistent with previously reported results. The formation of coated, or procoagulant platelets, has previously been linked with phenotype in hemophilia A. In this large single-center cohort, no association of procoagulant platelet formation with HSS was noted, either in the whole population, or when analyzed by genotype or prophylaxis versus on-demand therapy. Measures of thrombin generation in PRP have been weakly linked with phenotype in severe hemophilia A. In our single-center cohort, in severe hemophilia A patients with large genetic mutations a strong association was noted between HSS and both time to peak (r = .410) and lag time (r=.450). This association persisted in PPP for time to peak, but was lost in PPP for lag time demonstrating the critical modulatory role of platelets in this association. No significant association was noted with other measures of thrombin generation. The results presented here indicate a critical modulatory role for platelet-dependent initiation of thrombin generation in determining phenotypic severity in patients with severe hemophilia A. Confirmation of these results would identify a potentially early biologic signal that could be used to differentiate phenotypic severity in young patients with severe hemophilia A and suggest the potential utility of modulation of platelet function in severe hemophilia A patients, especially those with large genetic mutations. DisclosuresDunn:Biogen: Other: Unrestricted educational grant, Research Funding; Octapharma: Other: Unrestricted educational grant; NovoNordisk: Other: Unrestricted educational grant; Shire: Consultancy, Other: Unrestricted educational grant, Research Funding; Bayer: Consultancy, Other: Unrestricted educational grant; Alnylam: Other: Unrestricted educational grant; World Federation of Hemophilia USA: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Consultancy, Other: Unrestricted educational grant; Kedrion: Other: Unrestricted educational grant.

Platelet Dysfunction and Persistent Fibrinolysis in Pediatric Scoliosis Surgery Patients Receiving Tranexamic Acid

High blood loss requiring transfusion is a common complication in pediatric scoliosis surgery. Bleeding in patients undergoing spinal surgery is exacerbated by the development of coagulopathy in response to extensive activation of the acute phase response (APR). Coagulopathy is characterized by consumption of coagulation factors, inflammation, and hyperfibrinolysis. Although the administration of antifibrinolytics (e.g. tranexamic acid, TXA) has significantly reduced transfusions, patients still experience high rates of blood loss, suggesting either that TXA dosing needs optimization or that plasmin-independent aspects of coagulopathy could be driving bleeding. Major orthopedic surgeries are characterized by a reduction in platelet counts indicating the coagulopathic conditions of surgery exert a tropism on the circulating platelet population. Indeed, platelet dysfunction concomitant with trauma-induced coagulopathy has been extensively described in literature. Platelet dysfunction during adult orthopedic surgery has been observed but has not been correlated with bleeding, and platelet function in pediatric spine surgery has yet to be assessed. To determine if pediatric patients also experience platelet dysfunction, we measured platelet activation by whole blood flow cytometry during the course of scoliosis surgery in 8 pediatric patients. In parallel, circulating markers of the APR (i.e. IL-6, IL-10, IL-8), products of fibrinolysis (D-dimer), and indicators of consumptive coagulopathy (Protein C) were measured by multiplex ELISA. Samples were collected preoperatively (Preop), intraoperatively (every 2 hours, Intraop 1 and Intraop 2), immediately after closure (Postop) and the following morning (POD1). Samples were stimulated with submaximal concentrations of thrombin, convulxin (GPVI agonist, collagen receptor), and ADP. Platelet integrin activation was measured by PAC1 binding (GPIIbIIIa activation) and platelet secretion was measured by CD62p binding (P-selectin surface expression). We observed a progressive and statistically significant reduction in the platelet response (GPIIbIIIa activation and P-selectin expression) to both thrombin and convulxin stimulation during the procedure but with distinct temporal patterns. The reduction in response to thrombin became significant at Postop, remaining down even at POD1 (PAC1 max reduction, Postop 1: 32.5%, p=0.0403; P-selectin max reduction, POD1: 33.9%, p=0.016). The reduction in response to convulxin, on the other hand developed almost immediately after the initiation of surgery, starting with Intraop1 and resolving by Postop (PAC1 max reduction, Intraop 1: 25.1%, p=0.0087; P-selectin max reduction, Intraop 1: 18.3%, p=0.0369). Intra-operative responses to thrombin (both PAC1 and CD62p binding) significantly correlated with estimates of blood loss based on red blood cell mass (Estimated Red Blood Cell Mass, ERCM def) and hemoglobin mass (Hg mass loss) (see Table I below). Although we observed a consistent and statistically significant reduction in platelet number it correlated with blood loss at POD1 only, suggesting platelet function is a better predictor of blood loss than platelet count. Despite all patients receiving a bolus of TXA with continuous low dose infusion, multiplex results indicated a progressive increase in circulating D-dimer, becoming statistically significant at Postop and persisting through POD 1. Elevations in D-dimer, the byproduct of plasmin degradation of fibrin, suggest elusive fibrinolysis despite administration of TXA. Significant intraoperative elevations in IL-6, IL-10, and IL-8 and consumption of Protein C were also noted that persisted through POD1, indicating the development of coagulopathy. In conclusion, pediatric scoliosis surgery is characterized by persistent fibrinolysis platelet dysfunction, activation of the APR, and coagulopathy despite administration of the anti-fibrinolytic TXA. Future studies will focus on altering the dose of TXA to determine if an optimal dosing can be achieved that effectively inhibits fibrinolysis and answer important questions about the dependence of the APR and platelet hypofunction on the persistent fibrinolysis seen in these patients. Figure Disclosures No relevant conflicts of interest to declare.

D-Dimer and Fibrin Degradation Products Impair Platelet Signaling: Plasma D-Dimer Is a Predictor and Mediator of Platelet Dysfunction During Trauma.

Platelet dysfunction often accompanies trauma-induced coagulopathy. Because soluble fibrin impairs platelet glycoprotein VI (GPVI) signaling and platelets of trauma patients can display impaired calcium mobilization, we explored the role of fibrinolysis on platelet dysfunction during trauma. Convulxin-induced GPVI calcium mobilization was investigated in healthy platelet-rich plasma (PRP) pretreated with thrombin and tissue plasminogen activator (tPA). Blood samples from healthy participants (n = 7) and trauma patients (n = 22) were tested for platelet calcium mobilization, plasma D-dimer, platelet D-dimer binding (via flow cytometry), and platelet lumi-aggregometry. For healthy platelets, maximal platelet dysfunction was observed when cross-linked soluble fibrin (no tPA) or cross-linked fibrin degradation products (FDPs) were generated in suspension before convulxin stimulation. Lack of fibrin polymerization (inhibited by Gly-Pro-Arg-Pro [GPRP]) or lack of factor XIIIa cross-linking (T101-inhibited) restored GPVI signaling, whereas non-cross-linked FDPs only partially blocked signaling induced by convulxin. In addition, D-dimer added to healthy PRP impaired platelet aggregation and dense granule release induced by various agonists. Plasma D-dimer level was strongly correlated (R = 0.8236) with platelet dysfunction as measured by platelet calcium mobilization induced with various agonists. By 48 to 120 h after trauma, plasma D-dimer levels declined, and platelet function increased significantly but not to healthy levels. Trauma platelets displayed elevated D-dimer binding that was only partially reduced by αIIbβ3-inhibitor GR144053. After 60-minute incubation, washed healthy platelets resuspended in plasma from trauma patients captured approximately 10000 D-dimer equivalents per platelet. During trauma, D-dimer and FDPs inhibit platelets, potentially via GPVI and integrin αIIbβ3 engagement, contributing to a fibrinolysis-dependent platelet loss-of-function phenotype.

Honokiol as a specific collagen receptor glycoprotein VI antagonist on human platelets: Functional ex vivo and in vivo studies

Honokiol, derived from Magnolia officinalis, has various pharmacological properties. Platelet activation plays a critical role in cardiovascular diseases. Honokiol has been reported to inhibit collagen-stimulated rabbit platelet aggregation. However, detailed further studies on the characteristics and functional activity of honokiol in platelet activation are relatively lacking. In the present study, honokiol specifically inhibited platelet aggregation and Ca+2 ion mobilization stimulated with collagen or convulxin, an agonist of glycoprotein (GP) VI, but not with aggretin, an agonist of integrin α2β1. Honokiol also attenuated the phosphorylation of Lyn, PLCγ2, PKC, MAPKs, and Akt after convulxin stimulation. Honokiol have no cytotoxicity in zebrafish embryos. Honokiol diminished the binding of anti-GP VI (FITC-JAQ1) mAb to human platelets, and it also reduced the coimmunoprecipitation of GP VI-bound Lyn after convulxin stimulation. The surface plasmon resonance results revealed that honokiol binds directly to GP VI, with a KD of 289 μM. Platelet function analysis revealed that honokiol substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation in mice. In conclusion, honokiol acts as a potent antagonist of collagen GP VI in human platelets, and it has therapeutic potential in the prevention of the pathological thrombosis.

The Influence of FLI1 Expression Levels on Megakaryopoiesis: Studies Using iPSCs

Jacobsen syndrome is a rare, inherited hemizygous deletion of chromosome 11q that is often associated with a dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Among the genes involved in the chromosomal deletion are FLI1 and ETS1, both of which belong to the ETS family of transcription factors and have been associated with megakaryopoiesis. One prior study using primary human hematopoietic stem cells suggested that the defect in PTSx was due to FLI1 allelic exclusion resulting in the generation of two distinct megakaryocyte (MK) populations, one immature and one mature. More consistent with the clinical course of this disorder, we hypothesize that PTSx is caused by FLI1 haploinsufficiency, where all MKs are affected and do not mature properly. The goal of our studies was to better understand MK development by investigating the role of FLI1 during megakaryopoiesis, including in PTSx. However, Fli1 deletions in mice did not replicate the defect observed in humans, so we used genome-engineered human induced pluripotent stem cell (iPSC) lines. We have established an iPSC line from a PTSx patient and derived from this a FLI1 overexpressing (OE) line in which FLI1 cDNA was cloned into the AAVS1 "safe harbor" locus with MK-specific expression driven by the GP1bα promoter. In parallel, we have a healthy control line, a control-derived FLI1 OE line, and a homozygous FLI1+/- line. In the control- and PTSx-FLI1 OE lines, FLI1 mRNA levels in MKs were 2X higher than control levels. The FLI1+/- line was generated using TALENs and expressed RNA at levels comparable to the PTSx line. To analyze MK progenitor potential, the iPSC lines were differentiated to hematopoietic progenitor cells (HPCs) and analyzed using Megacult colony assays. The PTSx line generated 4- to 6-fold less CFU-MK colonies per 1000 plated CD41+CD235+ cells compared to control (P=0.1) and PTSx-FLI1 OE (P=0.002). Likewise, the FLI1+/- line had less colonies compared to control (P=0.2) and control-FLI1 OE (P=0.005). The control-FLI1 OE line generated 70% more colonies (P=0.22) than the control line. To analyze MK generation, identical numbers of HPCs were expanded in liquid culture containing MK-specific cytokines and the numbers of CD41+CD42a+ cells were quantitated. The PTSx line had <20% the number of MKs generated in the control. The numbers of MKs generated from the PTSx-FLI1 OE and FLI1+/- HPCs were about half that of control line, while the number of MKs generated from the control FLI1 OE line was 40% higher than control. Platelet function studies show that CD42b+ PTSx and FLI1+/- platelet-like particles (PLPs) were unresponsive to convulxin stimulation compared to CD42b+ control, control-FLI1 OE, and PTSx-FLI1 OE PLPs. In addition, the PTSx and FLI1+/- MKs began to lose CD42b after only 3 days in culture while the PTSx-FLI1 OE and control MKs began to lose CD42b after 6 days in culture. The control-FLI1 OE MKs still retained CD42b expression after 8 days in culture. Overall, these data support our hypothesis that FLI1 haploinsufficiency underlies PTSx, as two distinct MK populations were not observed. Furthermore, the FLI1+/- MKs had similar characteristics to the PTSx-derived MKs, which pinpoints FLI1 deletion as the cause of PTSx MK deficiency. More importantly, we show that MK commitment of HPCs, MK expansion and maturation, their ability to retain CD42b expression and response to agonist stimulation correlate with FLI1 expression levels. Our findings have implications for production of functional MKs and platelets for future clinical application. DisclosuresNo relevant conflicts of interest to declare.

Hinokitiol is a novel glycoprotein VI antagonist on human platelets

Hinokitiol (4-isopropyl-tropolone) is a bioactive compound with various pharmacological activities that is found in the wood of cupressaceous plants. Platelet activation plays an important role in thrombogenesis. In our previous study, hinokitiol specifically inhibited collagen-induced platelet aggregation ex vivo and prolonged thrombogenesis in vivo. The glycoprotein (GP) VI and integrin α2β1 are major collagen receptors that mediate platelet adhesion and aggregation. In our current study, we investigated which of these collagen receptors is involved in the hinokitiol-mediated inhibition of platelet activation. Treatment with 2–100 µM hinokitiol caused a dose-dependent right, parallel shift in the collagen concentration–response curve (0.5–10 µg/ml), with no change in the maximal responses. Furthermore, hinokitiol inhibited platelet aggregation and relative [Ca2+]i mobilization stimulated by convulxin, an agonist of GP VI, but not by aggretin, an agonist of integrin α2β1, indicating that hinokitiol mediates the inhibition of platelet activation through GP VI, rather than through integrin α2β1. Hinokitiol also specifically inhibited the convulxin-mediated activation of protein kinase C, phospholipase Cγ2, Akt, mitogen-activated protein kinases, and Lyn. Hinokitiol markedly diminished the co-immunoprecipitation of GP VI-bound Lyn after convulxin stimulation. In conclusion, hinokitiol, an antagonist of collagen GP VI may represent a novel antiplatelet drug for the prevention of thrombi associated with coronary and cerebral artery diseases.

Bruton's Tyrosine Kinase mediates platelet receptor-induced generation of microparticles: A potential mechanism for amplification of inflammatory responses in rheumatoid arthritis synovial joints

Platelet microparticles (pMPs) are small membrane-coated vesicles that are released from the plasma membrane upon platelet activation. In the joint fluid of patients with rheumatoid arthritis, pMP can interact with and activate fibroblast-like synoviocytes (FLS), which are important effector cells that mediate both immune activation and joint destruction. The signaling process by which engagement of glycoprotein VI (GPVI), a surface glycoprotein receptor for collagen which is expressed on platelets, triggers pMP generation is poorly understood, but has been suggested to involve Spleen Tyrosine Kinase (SYK), best known as an upstream activator of Bruton's Tyrosine Kinase (BTK) in B cells. In this study, we showed that activation of human platelets triggered by convulxin or collagen, specific ligands for GPVI receptor, or alternatively by antibody-mediated cross-linking of another platelet receptor, C type lectin-like receptor 2 (CLEC2), resulted in phosphorylation of BTK and downstream effector, phospholipase Cγ2 (PLCγ2). A potent and selective BTK inhibitor, RN486, inhibited GPVI- or CLEC2-mediated PLCγ2 phosphorylation and pMP production in a dose-dependent manner. BTK is also an essential effector of B cell receptor (BCR)-induced B cell signaling. Consistent with the biology, the IC50s of BTK inhibitors with varying potencies in a BCR-dependent B cell activation marker assay correlated with those in the GPVI-mediated PLCγ2 phosphorylation. In a co-culture system consisting of human primary synovial FLS and activated human platelets, convulxin stimulation resulted in elevated production of pro-inflammatory cytokines, IL-6 and IL-8, an effect which was dose-dependently blocked by RN486. The effects are specific as RN486 abrogated platelet aggregation induced by GPVI ligands but not by other platelet surface receptor agonists. Taken together, our data further support the potential therapeutic utility of BTK inhibitors in RA therapy, by inhibiting GPVI-mediated platelet activation and thus subsequent amplification of inflammation driven by pMP-induced FLS cytokines production.

Over-Expression of the Mas Receptor Decreases Arterial Thrombosis Risk in B2R KO Mice by Elevating NO and Prostacyclin and Reducing GPVI Activation

Abstract 700 Background:Bradykinin B2 receptor KO mice (B2R KO) were recognized to have delayed rose bengal and ferric chloride carotid artery thrombosis times and long tail bleeding times. In B2R KO, elevated serum angiotensin II (AngII) binds to an over-expressed angiotensin receptor 2 (AT2R) to increase plasma NO and prostacyclin (Blood 108:192, 2006). It was proposed that the combined presence of elevated AngII and AT2R are essential for the thrombosis protection phenotype. However, after losartan treatment, an angiotensin receptor 1 antagonist, thrombosis remains delayed in B2R KO despite reduction in AngII levels from 258±64 to 40±15 pg/ml. An additional pathway for thrombosis protection in B2R KO mice was sought. Methods and Results:Losartan treatment lowers ACE mRNA in mice along with lowering AngII levels. However, even in the presence of low AngII levels, plasma Ang1-7 levels, the breakdown product of AngII, in losartan-treated B2R KO are increased [21±2.2 pg/ml vs 14.4±0.7 pg/ml in wild type mice (WT)]. Upon examination, B2R KO also have elevated Mas mRNA, the receptor for angiotensin1-7, with over-expressed renal Mas protein. Treatment of B2R KO with A-779, a Mas antagonist, shortens carotid artery thrombosis time (58±4 to 38±4 min) and tail bleeding time (170±13 to 88±8 sec) and lowers plasma concentrations of nitrate (from 21.5±3.6 micromolar in untreated B2R KO to 15±5 micromolar in A-779-treated mice) and 6-keto-PGF1alpha (259±103 pg/ml in untreated B2R KO to 132±58 pg/ml). ADP- or thrombin-induced platelet (plt) activation are not abnormal in B2R KO, either by aggregation studies or by flow cytometric studies examining for fibrinogen binding or JON/A integrin epitope binding and P-Selectin expression, respectively. Although static and flow adherence to collagen was normal, B2R KO plts have defective spreading on collagen (20±0.6 microns in B2R KO versus 27±0.4 microns in WT, p<0.0001) under static conditions. Resting B2R KO plts constitutively also have increased DAF-FM fluorescence (649±41 AFU versus 405±36 AFU, p=0.0012), suggesting increased plt NO without stimulation. GSNO, an NO donor, or carbaprostacyclin, a stable derivative of prostacyclin, inhibits murine plt spreading on collagen in a concentration-dependent manner. B2R KO plts have reduced GPVI activation using CRP or convulxin even though GPVI expression on B2R KO and WT plts are similar. Further, B2R KO plts have reduced Syk phosphorylation after convulxin stimulation and reduced phosphorylation of Src family kinases when spreading on collagen. Treatment of B2R KO plts in vivo with A-779 corrects the spreading and Src family kinase phosphorylation defects upon collagen exposure, suggesting an in vivo role of the Mas receptor in the plt function defect and thrombosis delay. Conclusion:B2R KO are protected from thrombosis by over-expression of two receptors (AT2R and Mas) from the renin-angiotensin system resulting in increased NO and prostacyclin and an acquired platelet function defect. The receptors AT2R and MAS compensate for the absence of the B2R. These studies show novel mechanism(s) whereby receptors in the renin-angiotensin system influence platelet function and arterial thrombosis risk in vivo. Disclosures:No relevant conflicts of interest to declare.

The human megakaryocytic cell line UT‐7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor

We have demonstrated that a unique megakaryocytic cell line UT-7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT-7/TPO was found to express GPVI and its associate signal molecule of FcRgamma (Fc receptor gamma chain). When UT-7/TPO was stimulated with the GPVI agonist convulxin, the [Ca2+]i (intracellular Ca2+) was elevated in a convulxin concentration-dependent manner, and [Ca2+]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT-7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)-1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca2+]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin-stimulated platelets failed to produce TX, it is suggested that UT-7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT-7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor γ-chain (FcR γ-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca++ elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase Cγ2 (PLCγ2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca++]i, during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR γ-chain during differentiation of megakaryocytes.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

AbstractThis study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor γ-chain (FcR γ-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca++ elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase Cγ2 (PLCγ2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca++]i, during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR γ-chain during differentiation of megakaryocytes.

Electron microscopic cytochemistry on the distribution of wheat germ agglutinin receptor on the platelet plasma membrane after treatment with convulxin isolated from Crotalus durissus terrificus venom

The effects of convulxin, isolated from the venom of Crotalus durissus terrificus, on the localization and distribution of wheat germ agglutinin (WGA) binding sites on platelet surfaces have been investigated at an ultrastructural level. A post-embedding cytochemical technique using WGA-gold complexes was used and the quantitative intensity of WGA-labeling on the surface membrane of platelets after convulxin stimulation was determined. In the presence of Ca 2+, convulxin induced platelet release and aggregation, while in its absence, the platelets formed pseudopodia and showed release reaction, but without aggregation. After treatment with convulxin, WGA-labeling on the surface membrane decreased compared with intact washed (control) platelets. In the presence of Ca 2+, clusters of gold label were often found on the surface membrane. However, the WGA-labeling intensity of the membrane of the open canalicular system increased significantly compared with that of platelets stimulated by convulxin in the absence of Ca 2+. Direct morphological evidence demonstrates qualitative and quantitative alterations of WGA-labeling on the surface membrane of platelets, after convulxin stimulation. The possibility is considered that WGA-binding glycoproteins on the surface membrane are involved in the aggregation response after convulxin stimulation.