ADP-coupled integrin regulation by the extracellular redox system.
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Adenosine Diphosphate↗
Biomedical subjects
B Kehrel
Publications and source records attributed to B Kehrel.
P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo.
The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin-immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin-bound ligands were eluted with ethylenediaminetetraacetic acid. An approximately 210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti-human PSGL-1 antibody and could be blotted with P-selectin-IgG. Under reducing conditions, both the predicted PSGL-1 approximately 210-kD dimer and the approximately 120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25-100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti-PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.
Animals↗
The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function.
The vasodilator-stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP- and cGMP-dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP-deficient mice had normal agonist-induced contraction, and normal cAMP- and cGMP-dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP- and cGMP-mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP- and cGMP-dependent effects in platelets, such as inhibition of agonist-induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide-dependent mechanisms are operating during platelet activation: a VASP-independent mechanism for inhibition of calcium mobilization and granule release and a VASP-dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.
Animals↗
New strategies in diagnosis and treatment of thrombotic thrombocytopenic purpura: case report and review.
UNLABELLED: The pentad of thrombocytopenia, haemolytic anaemia, mild renal dysfunction, neurological signs and fever, classically characterizes the syndrome of thrombotic thrombocytopenic purpura (TTP). TTP usually occurs in adults but also children have been described with this condition. The disorder may take a relapsing course, termed chronic relapsing TTP (CRTTP), which although very rare, may also begin in childhood. Deficiency of a recently identified enzyme, the von Willebrand factor (vWF)-cleaving protease, seems to play a major role in the development of TTP. We report on a 3-year-old boy with a dramatic but typical clinical course of CRTTP. At the time of diagnosis, neurological deficits and multiple cerebral infarctions had already occurred. In plasma, vWF-cleaving protease was completely absent, both during acute TTP and in remission. There was no protease inhibitor detected. Regular infusions of fresh frozen plasma were successfully given for replacement on a prophylactic basis. CONCLUSION: Assay of von Willebrand factor-cleaving protease helps to diagnose a form of thrombotic thrombocytopenic purpura which may be managed by prophylactic treatment with fresh frozen plasma.
Blood Coagulation Tests↗
Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not.
Simple collagen-related peptides (CRPs) containing a repeat Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events induced by the peptides are the same as most of those induced by collagen. The peptides do not recognize the alpha 2 beta 1 integrin. To identify the signaling receptor involved, we have evaluated the response to the CRP, Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets with defined functional deficiencies. These studies exclude a primary recognition role for CD36, von Willebrand factor (vWF), or glycoprotein (GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited normal aggregation, normal fibrinogen binding, and normal expression of CD62 and CD63, measured by flow cytometry, in response to the peptide, and there was normal expression of CD62 and CD63 on thrombasthenic platelets. In contrast, GPVI-deficient platelets were totally unresponsive to the peptide, indicating that this receptor recognizes the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed some fibrinogen binding in response to collagen but failed to aggregate and to express CD62 and CD63. Collagen, but not CRP-XL, contains binding sites for alpha 2 beta 1. Therefore, it is possible that collagen still induces some signaling via alpha 2 beta 1, leading to activation of GPIIb/IIIa. Our findings are consistent with a two-site, two-step model of collagen interaction with platelets involving recognition of specific sequences in collagen by an adhesive receptor such as alpha 2 beta 1 to arrest platelets under flow and subsequent recognition of another specific collagen sequence by an activatory receptor, namely GPVI.
Binding Sites↗
Flow cytometric analysis of agonist-induced annexin V, factor Va and factor Xa binding to human platelets.
Activated platelets provide a procoagulant surface for the assembly and expression of prothrombinase complex. Expression of activity is associated with the binding of the protease factor Xa (FXa) and the co-factor Va (FVa) to the procoagulant surface. A flow cytometric methodology to measure annexin V-FITC as well as FVa and FXa binding to ionophore A 23187 activated platelets is described. Annexin V-FITC was used to determine platelet exposure of phosphatidylserine. The binding was calcium-dependent and excess of unlabelled annexin V (10-fold) prevented the binding of the labelled protein. The binding of FVa and FXa to platelets was measured using specific FITC-labelled monoclonal antibodies. The FITC labelled antibodies were displaced by 10-to 20-fold excess of unlabelled antibodies. Binding was strictly Ca2+-dependent. Fixation of platelets by formaldehyde caused artificial binding of annexin V, FVa and FXa as well, irrespective of the platelet activation status. Using gel-filtered platelets, the binding of FVa increased with alpha -granule secretion but the amount of stored FVa was not sufficient to saturate the available platelet binding sites. Exogenous FVa was needed for maximal FVa binding to occur. No binding of FXa from internal platelet stores was observed. Addition of exogenous FVa and FXa resulted in FXa binding to the platelet surface. The methodology might be of use for the study of platelets from patients with bleeding disorders.
Journal Article↗
HDL3-mediated inhibition of thrombin-induced platelet aggregation and fibrinogen binding occurs via decreased production of phosphoinositide-derived second messengers 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate.
We demonstrate that physiological concentrations of HDL3 inhibit the thrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL3-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate turnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation, and intracellular calcium mobilization. The inhibitory effects of HDL3 on inositol 1,4,5-tris-phosphate formation and intracellular calcium mobilization were abolished after covalent modification of HDL3 with dimethylsuberimidate. Furthermore, they could be blocked by calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated protein kinase C inhibitors. However, the inhibitory effects of HDL3 were not blocked by H89, a protein kinase A inhibitor. In addition, HDL3 failed to induce cAMP formation but stimulated the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL3-mediated inhibition of thrombin-induced inositol 1,4,5-tris-phosphate formation, intracellular calcium mobilization, and fibrinogen binding and the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. Taken together, these findings indicate that the HDL3-mediated inhibition of thrombin-induced fibrinogen binding and aggregation occurs via inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and formation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Protein kinase C may be involved in this process.
Adult↗
Human platelet CD36 (GPIIIb, GPIV) binds to cholesteryl-hemisuccinate and can be purified by a simple two-step method making use of this property.
CD36, also known as GPIIIb or GPIV, is a main protein of the platelet membrane. Accumulating evidence implicates CD36 in the pathogenesis of atherosclerosis. CD36 binds to cholesteryl-hemisuccinate coupled to agarose. This binding may be of physiologic relevance since CD36 has a long extracellular hydrophobic region at its N-terminus and belongs to a family of proteins involved in lipid metabolism. This newly discovered binding property of CD36 was used to develop a rapid, efficient and gentle two-step purification method for CD36 from human platelets.
Blood Platelets↗
Low-density lipoproteins inhibit the Na+/H+ antiport in human platelets. A novel mechanism enhancing platelet activity in hypercholesterolemia.
BACKGROUND: LDL have been reported to augment platelet activation, and increased platelet reactivity has been observed in familial hypercholesterolemia. However, the underlying mechanisms of this putatively atherogenic effect is unknown. Because intracellular pH (pHi) may play an important role in platelet function, we examined the influence of LDL on pHi and Na+/H+ antiport activity in human platelets and compared it with the effect of [3-methylsulfonyl-4-piperidinobenzoyl] guanidine hydrochloride (HOE 694), a selective Na+/H+ antiport inhibitor. METHODS AND RESULTS: Using a fluorescent dye technique, we demonstrated that incubation of platelets with physiological concentrations of LDL or with HOE 694 decreased pHi. In addition, both LDL and HOE 694 inhibited the Na+/H+ antiport in platelets treated with sodium propionate or thrombin. The inhibitory effect of LDL was observed both in normal and in glycoprotein (GP)IIb/IIIa-as well as in GPIIIb (CD36)-deficient platelets and was not influenced by the covalent modification of apolipoprotein B lysine residues, suggesting that specific LDL binding sites were not involved. Thrombin-induced phosphoinositide breakdown, diacylglycerol formation, and Ca2+ mobilization, as well as platelet aggregation and granule secretion, were potentiated by both LDL and HOE 694. pHi and Na+/H+ antiport activity were significantly reduced in platelets from patients with familial hypercholesterolemia. Both parameters were normalized after normalization of LDL levels by apheresis treatment. CONCLUSIONS: LDL inhibits the Na+/H+ antiport most likely via receptor-independent mechanisms, thereby augmenting platelet reactivity. This novel mechanisms explains increased platelet reactivity in patients with familial hypercholesterolemia and may contribute to the atherogenic potential of LDL.
Adult↗
Formation of platelet-leukocyte conjugates in whole blood.
The purpose of this investigation was to obtain information on platelet-leukocyte conjugate formation in whole blood and on factors that affect it. We also measured platelet and leukocyte activation by quantitating the expression of CD62P and CD11b. In both cases a flow cytometric approach was used. The results show that platelet-monocyte and platelet-polymorphonuclear leukocyte (PMNL) conjugate formation is enhanced by simply stirring blood, with optimum conjugate formation occurring after 10 min. In the case of monocytes,conjugate formation was enhanced by adenosine diphosphate (ADP). Both monocyte and PMNL conjugate formation was enhanced by phorbol myristate acetate (PMA), but L-formyl methionyl lysyl proline (FMLP) was either without effect (monocytes) or inhibitory (PMNL). EDTA also inhibited conjugate formation (implying involvement of divalent cations), as did dextran sulphate (implying involvement of P-selectin = CD62P). Interestingly GR144053F, which acts at GpIIb-IIIa on platelets to interfere with fibrinogen binding, and also glycyl prolyl arginyl proline (GPRP), a peptide that interferes with the interaction between CD11c on leukocytes and fibrinogen, did not inhibit platelet-monocyte conjugate formation, but did inhibit the platelet-PMNL interaction; this indicates that GpIIb-IIIa on platelets and CD11c on leukocytes and fibrinogen are involved in mediating the interaction between platelets and PMNL but not platelets and monocytes. Surprisingly arginyl-glycyl aspartyl serine (RGDS) inhibited the formation of both types of conjugate but this may be because it also inhibited both platelet and leukocyte activation as measured by CD62P and CD11b exposure and/or interferes with the binding of adhesion molecules other than fibrinogen. The results show that a flow cytometric procedure can be effective in obtaining rapid information on platelet-leukocyte conjugate formation in whole blood and on factors that are involved in its regulation. It is suggested that the technique may be applicable to the study of platelet-leukocyte conjugate formation in whole blood in disease, and also to study the effects of drugs interfering with conjugate formation.
Journal Article↗
Interaction of von Willebrand factor with Staphylococcus aureus.
Intravascular infection due to Staphylococcus aureus requires colonization of subendothelium in the presence of shear forces. von Willebrand factor (VWF) is a large multimeric glycoprotein playing a key role in platelet adhesion to subendothelium. To determine whether VWF may also play a role in adhesion of S. aureus to endovascular sites, binding of VWF to S. aureus and adhesion of S. aureus to VWF-adsorbed substrates was examined. Binding isotherms revealed a dose-dependent reaction of purified VWF with S. aureus Cowan 1 as well as VWF binding to other S. aureus strains. On solid phase, VWF showed saturable adsorption kinetics to polymethylmethacrylate and promoted S. aureus adhesion up to 67-fold in a trypsin-sensitive reaction. Similar adhesion promotion was observed when recombinant VWF was used. These results show that VWF interacts with S. aureus in suspension and promotes S. aureus adhesion to surfaces, suggesting a role of VWF in the pathogenesis of intravascular S. aureus infections.
Bacterial Adhesion↗
Arg506 to Gln mutation in the factor V gene causes poor fibrinolytic response in children after venous occlusion.
To determine to what extent the Arg506 to Gln mutation in the factor V gene influences the fibrinolytic response after 20 min venous occlusion (VO) we investigated a population of APC resistant children (n = 60) and a group of age-matched healthy controls (n = 25). After 20 min VO, symptomatic (n = 30) carriers of the common factor V mutation showed significantly reduced t-PA activities compared with asymptomatic (n = 30) carriers (p <0.0001) and healthy controls (p <0.0001). In contrast, PAI 1 activity was significantly (p <0.0001) higher before and after VO in children with the factor V mutation compared with healthy children. No difference was found between symptomatic and asymptomatic probands. A significantly lower PAI 1 antigen decrease along with a lower t-PA antigen release was found in the APC resistant children compared with the controls. No significant difference was seen between individuals with and without previous vascular insults. As the lack of t-PA activity after VO in symptomatic carriers is the most conspicuous result, we suggest that the factor V gene mutation itself might induce the fibrinolytic impairment by increasing the thrombin levels and thus increasing the recently described thrombin-activable fibrinolysis inhibitor (TAFI).
Arginine↗
Flush heparin during cardiac catheterisation prevents long-term coagulation activation in children without APC-resistance-preliminary results.
This study was designed to prospectively evaluate haemostatic activation in 75 children undergoing cardiac catheterisation with intermittent flush heparin (10 IU/ml saline) and to relate these data to clinical findings and inherited risk factors for thrombophilia. In addition to flush heparin in infants < 6 months of age in whom additional arterial catheterisation was performed (n = 5) or patients with thrombophilia, heparin (300-400 IU/kg/d) was administered for a further 24 h. APTT was prolonged and anti Xa activity was significantly increased at the end of catheterisation and returned to normal 24 hours later. Whereas thrombin generation (F1 + 2) showed a significant coagulation activation at the end of catheterisation, no concomitant fibrinolytic activation (D-Dimer) was observed. Four children showed resistance to APC: one of them in whom stroke had occurred before and one additional child heterozygous for APCR received further prophylactic heparin. Two neonates with APCR and flush heparin only suffered from thrombosis after catheterisation. No further thrombotic events occurred. This study indicates that low-dose flush heparin during catheterisation may prevent long-term haemostatic activation in children without thrombophilia. Whether further heparin after cardiac catheterisation in children with APCR prevents vascular insults requires a more intensive study.
Adolescent↗
Thrombospondin measured in whole blood--an indicator of platelet activation.
Abnormal platelet activation may be involved in prethrombotic states and lead to thromboembolism. When platelets become activated, they release thrombospondin (TSP) from their alpha-granules which binds mainly to the surface of activated platelets, platelet-derived microparticles and other blood cells. To determine bound as well as free TSP in a single assay, we developed an indirect ELISA to measure TSP in fixed whole blood. The intra-assay variance was less than 5% and 97% of purified standard TSP, added to whole blood samples, was recovered with the ELISA. Blood collected with a 20G needle into a syringe resulted in lower "whole blood TSP' values than blood collected with the Vacutainer system. Whole blood TSP levels were measured in 66 healthy blood donors (20F, 46M) aged 25-75 years. The mean whole blood TSP concentration was 33 +/- 19 ng/ml. No significant difference in whole blood TSP was found between healthy females and males (35 +/- 23 ng/ml vs. 33 +/- 17 ng/ml).
Adult↗
High density lipoproteins enhance the Na+/H+ antiport in human platelets.
In the present study, we investigated the effect of high density lipoproteins 3 (HDL3) on Na+/H+ exchanger activity and cytosolic pH (pHi) in human platelets. HDL3 alone failed to affect pHi, but preincubation with HDL3 significantly enhanced the Na+/H+ antiport activation brought about by acidification with 100 mM sodium propionate or stimulation with 0.05 U/ml thrombin. the stimulatory effect of HDL3 was unaffected by indomethacin excluding a role for cyclooxygenase products. The HDL3 effect was not mediated by Ca2+/calmodulin-dependent protein kinase as HDL3 failed to increase cytosolic free calcium concentration. However, the potentiating effect of HDL3 was completely blocked in the presence of the protein kinase C inhibitor, bisindoylmaleimide and the phosphatidylcholine-specific phospholipase C inhibitor, D609. Furthermore, the effect of HDL3 was abolished after covalent modification of HDL3 with dimethylsuberimidate and was not observed in platelets from Glanzmann thrombasthenia type 1 which do not express GP IIb/IIIa, as well as in platelets preincubated with anti-GP IIb/IIIa polyclonal antibodies. We conclude that HDL3 enhances the sodium propionate- and thrombin-induced Na+/H+ antiport activity in human platelets via binding to GP IIb/IIIa and activation of protein kinase C and phosphatidylcholine-specific phospholipase C.
Blood Platelets↗
HDL3 activates phospholipase D in normal but not in glycoprotein IIb/IIIa-deficient platelets.
Glycoprotein IIb/IIIa has been proposed as the platelet receptor for high density lipoproteins (HDL3). We characterized the HDL3-induced second messenger response in normal and glycoprotein IIb/IIIa-deficient platelets. In normal platelets physiological concentrations of HDL3 induced the time-dependent generation of phosphatidic acid in the absence of phosphoinositide turnover. The rise in phosphatidic acid preceded that of diacyglycerol which was inconsistent with phospholipase C/diacylglycerol kinase pathway being the source of phosphatidic acid and suggested the involvement of phospholipase D. In the presence of butanol, HDL3 stimulated the accumulation of phosphatidylbutanol, an unequivocal indicator of phospholipase D activity. No increase in phosphatidic acid, diacylglycerol, and phosphatidylbutanol was observed upon addition of HDL3 to glycoprotein IIb/IIIa-deficient platelets. We conclude that phosphatidic acid is generated in HDL3-stimulated platelets by phospholipase D and that glycoprotein IIb/IIIa is the receptor involved in this process.
Diglycerides↗
Acquired von Willebrand disease in malignant peripheral neuroectodermal tumor (PNET).
In week 12 of the EICESS 92 protocol a 12-year-old boy with pelvic PNET developed acquired von Willebrand disease: bleeding time was prolonged (> 15 min) and von Willebrand factor antigen (54%) and ristocetin cofactor activity (50) were reduced. Platelet aggregations with thrombin and collagen and the number of major platelet glycoproteins/per platelet did not differ from the controls. After Haemate P (Behring Werke, Marburg, Germany) bleeding time normalized and surgery could be performed without occurrence of bleeding episodes. Three weeks after surgery bleeding time, ristocetin cofactor activity, and von Willebrand factor antigen increased to normal paediatric values. Without occurrence of further bleeding episodes the patient received another 10 courses of polychemotherapy (EICESS 92).
Bone Neoplasms↗
Platelet-collagen interactions.
The collagens belong to the constituents of the subendothelium that determine the thrombogenicity of the vessel wall. The existence of several genetically distinct collagens is well documented. To date, 19 collagens have been characterized. The collagens are divided into the fibril forming collagens and the non-fibril forming collagens. At least nine of the different collagens--type I, III, IV, V, VI, VIII, XII, XIII, XIV--were found in the vessel wall. All collagen molecules are built of three chains in a triple-helical conformation. Some collagens also contain large parts of non-collagenous domains. An interesting example for complex collagens is collagen type VI for it shares non-collagenous domains with the von Willebrand factor A domain, the platelet glycoprotein Ib, fibronectin type III repeats, and Kunitz type protease inhibitor. Monomeric and fibrillar collagens effectively support platelet adhesion, whereas for collagen-induced platelet aggregation and secretion, the native, triple-helical structure of collagen is required. The platelet reactive sites in collagens type I and III have been studied intensively. Using CNBr-peptides three aggregatory sites in the alpha 1(I) chain and one in the alpha 1(III) chain have been found. Cyanogen bromide fragments of collagen type I were also used to measure platelet adhesion under flow conditions. alpha 1(I)CB3 strongly supports platelet adhesion. The two peptides alpha 1(I)CB3 and alpha 1(III)CB4 are highly homologous. alpha 1(III)CB4 is highly aggregatory. Several substances are known to interfere at different levels with the platelet-collagen interactions and have been identified in blood-feeding animals. Among them are the "leech anti-platelet protein" (LAPP), calin, moubatin, and pallidipin.
Animals↗