Reversibility of platelet P2Y12 inhibition by platelet supplementation: ex vivo and in vitro comparisons of prasugrel, clopidogrel and ticagrelor.

Essentials Successful outcome of platelet transfusion depends on specific antiplatelet therapy in use. We assessed if ticagrelor, clopidogrel or prasugrel impacts on donor platelet activity ex vivo. Ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets. This might compromise the effectiveness of platelet transfusion therapy. SUMMARY: Background Platelet transfusion is the conventional approach to restore platelet function during acute bleeds or surgery, but successful outcome depends on the specific antiplatelet therapy. Notably ticagrelor is associated with inadequate recovery of platelet function after platelet transfusion. We examined whether plasma and/or platelets from ticagrelor-treated patients influence donor platelet function, in comparison with clopidogrel and prasugrel. Methods Platelet transfusion was mimicked ex vivo by mixing naïve donor platelet-rich plasma (PRP) or gel-filtered platelets (GFP) in defined proportions with PRP, plasma or GFP from cardiovascular patients receiving standard care including medication with prasugrel, clopidogrel or ticagrelor (n = 20 each). Blood was taken 4 h after the previous dose. HLA2/HLA28 haplotyping let us distinguish net (all platelet) and individual patient/donor platelet reactivity in mixtures of patient/donor platelets, measured by flow cytometry analysis of ADP-induced fibrinogen binding and CD62P expression. Results ADP responsiveness of donor platelets was dramatically reduced by even low (10%) concentrations of PRP or plasma from ticagrelor-treated patients. Clopidogrel and prasugrel were associated with more modest donor platelet inhibition. GFP from ticagrelor-treated patients but not patients receiving clopidogrel or prasugrel also suppressed donor GFP function upon mixing, suggesting the transfer of ticagrelor from patient platelets to donor platelets. This transfer did not lead to recovery of ADP responsiveness of patient's platelets. Conclusion Collectively, these observations support the concept that ticagrelor and/or its active metabolite in plasma or bound to platelets can inhibit donor platelets, which might compromise the effectiveness of platelet transfusion therapy.

[Pathophysiology and biochemistry of platelets]. / Pathophysiologie und Biochemie der Thrombozyten.

Although their central role is to control bleeding and to induce thrombosis, platelets are important inflammatory and immune cells as well as modulators of angiogenesis. This review focuses on the different roles of platelets in hemostasis, thrombosis, inflammation, arteriosclerosis, angiogenesis, antimicrobial host defense and hematogenous tumor metastasis. Platelets are the central regulators of hemostasis. On their surface the important thrombin burst takes place. Platelets cause atherothrombotic vascular occlusions. However, they are probably involved in early stages of arteriosclerosis, e.g. extravasation of leukocytes at sites of vascular injury, formation of foam cells and proliferation of smooth muscle cells. These processes are triggered by secretion of proinflammatory substances and growth factors as well as by platelet-cell interactions via specific adhesive axes. During infections platelets kill pathogens through secretion of antimicrobial substances and extracellular traps or nets. Platelets facilitate the revascularisation of ischemic tissue and therefore even promote tumor growth.

Anticoagulative management of patients requiring left ventricular assist device implantation and suffering from heparin-induced thrombocytopenia type II.

BACKGROUND: Heparin-induced thrombocytopenia type II (HIT II) is a rare but life-threatening side effect of heparin therapy. We describe the perioperative anticoagulative management of patients tested positive for HIT II and requiring implantation of a left ventricular assist device (LVAD). METHODS: We report on 3 patients with a different perioperative anticoagulative management (preoperative, intraoperative, and postoperative anticoagulation with danaparoid-sodium; preoperative anticoagulation with recombinant hirudin, anticoagulation with danaparoid-sodium intraoperatively and postoperatively; preoperative anticoagulation with recombinant hirudin, intraoperative anticoagulation with heparin, and postoperative anticoagulation with danaparoid-sodium) and discuss the difficulties of the treatment. RESULTS: Anticoagulation with alternative drugs such as recombinant hirudin and danaparoid-sodium led to serious and life-threatening bleeding complications as well as to thromboembolic events in the first 2 patients. Therefore the third patient underwent LVAD implantation using heparin for intraoperative anticoagulation to avoid administration of high doses of recombinant hirudin or danaparoid-sodium. Despite very low anti-factor Xa activities, when using danaparoid-sodium postoperatively, the patient suffered from a bleeding complication on the 4th day after LVAD implantation requiring reexploration. CONCLUSIONS: In selected cases (negative heparin-induced platelet aggregation (HIPA) test at the time of LVAD implantation and continuation of postoperative anticoagulation with recombinant hirudin or danaparoid-sodium), heparin may be used for LVAD implantation in HIT II patients to reduce bleeding complications.

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.

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.

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.

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.

Inhibition of hypercoagulation by antithrombin substitution in E. coli L-asparaginase-treated children.

Acquired deficiency of antithrombin (AT), which in some patients could lead to thrombosis, has been a serious side effect of protocols which incorporate E. coli L-asparaginase (ASP) for the treatment of acute lymphoblastic leukaemia (ALL). In a longitudinal, prospective, non-randomized study children with ALL (n=27) were treated according to the protocol ALL-BFM-90. During the induction phase using prednisone, vincristine, daunorubicin and ASP, AT substitution was performed in 15/27 patients, when their plasma concentration decreased below 60% of normal with a concomitant increase of D-dimer formation. After the administration of the AT concentrate the patients, plasma concentration of AT increased and remained elevated after 18, 48, and 72 h. In addition, the plasma concentration of enhanced thrombin generation, D-dimer formation and plasminogen activator inhibitor 1 decreased towards normal levels. Although the observed laboratory findings may serve as evidence for a possible clinical benefit of AT substitution during ASP treatment, further randomized studies are requested to evaluate whether the use of prophylactic AT administration could reduce the incidence of thromboembolic events in childhood acute leukaemia.

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).

Platelets deficient in glycoprotein IIIb aggregate normally to collagens type I and III but not to collagen type V.

The aggregation of platelets induced by collagens is considered an important step in primary hemostasis. Glycoprotein (GP) IIIb (GPIIIb, GPIV, CD36) has been proposed as a blood platelet receptor for collagen. Platelets from three healthy blood donors were shown to be clearly deficient in GPIIIb. These platelets aggregated normally in response to type I and III collagens. In addition, platelet factor 4, beta-thromboglobulin, and adenosine triphosphate (ATP) secretion in response to type I and III collagens was normal. The findings indicate that GPIIIb is not the major, essential collagen receptor for type I and III collagens. This would explain why all individuals with GPIIIb-deficient platelets examined so far are healthy and, in particular, show no apparent evidence of hemostatic problems. However, in contrast to control platelets, no aggregation and impaired platelet factor 4, beta-thromboglobulin, and ATP secretion was observed in response to type V collagen. Therefore, it is postulated that for type V collagen-induced aggregation both GPIa/IIa and GPIIIb are essential.

Role of platelet membrane glycoproteins Ib/IX and IIb/IIIa, and of platelet alpha-granule proteins in platelet aggregation induced by human osteosarcoma cells.

We have previously shown that the platelet-aggregating activity of human MG-63 and HOS osteosarcoma cells depends at least in part upon tumor cell surface-associated thrombospondin, and suggested that platelet-osteosarcoma cell interactions could occur through interactions with specific platelet membrane receptors. In this study, the platelet-aggregating activity of MG-63 and HOS cells was studied by using a variety of platelet disorders. Both osteosarcoma cell lines induced a biphasic platelet aggregation response when added to normal platelet-rich plasma, while the second phase of aggregation was absent when added to gray platelets (deficiency in alpha-granule proteins) and to aspirin-treated platelets. Platelets from two unrelated patients with type I Glanzmann's thrombasthenia (deficiency in glycoprotein (GP) GPIIb/IIIa) did not aggregate at all with osteosarcoma cells. Using giant platelets from three patients with Bernard-Soulier syndrome (deficiency in GPIb/IX), the aggregation response induced by MG-63 and HOS cells was monophasic and reversible when compared to normal-sized platelets and to giant platelets from a patient with May-Hegglin anomaly (no membrane GP defect). Because GPIb serves as a receptor for von Willebrand factor during hemostasis, aggregation experiments were also conducted with the platelet-rich plasma of two patients with a low plasma von Willebrand factor concentration (type I von Willebrand's disease) before and after the infusion of deamino-D-arginine vasopressin. MG-63 and HOS cells induced biphasic platelet aggregation both before and after deamino-D-arginine vasopressin treatment, while the ristocetin-dependent binding of von Willebrand factor to platelets only occurred after deamino-D-arginine vasopressin treatment. Preincubation of normal platelet-rich plasma with monoclonal antibody SZ-2 directed against the von Willebrand binding domain of GPIb did not inhibit the platelet-aggregation activity of osteosarcoma cells, whereas anti-GPIb antibody SZ-2 did inhibit ristocetin-induced platelet agglutination. In addition, anti-GPIX antibodies did not affect platelet-osteosarcoma cell interactions. In conclusion, our data demonstrate that the first phase of the platelet-aggregating activity of human osteosarcoma cells is initiated by the interaction of these tumor cells with platelet membrane GPIIb/IIIa, whereas the second phase, even if plasma von Willebrand factor is deficient, involves platelet membrane GPIb and the participation of platelet alpha-granule proteins in membrane-mediated events, making aggregation irreversible.

Platelet membrane activation markers are predictive for increased risk of acute ischemic events after PTCA.

BACKGROUND: We wished to investigate whether platelet activation is related to the clinical outcome during the 24 hours immediately after elective percutaneous transluminal coronary angioplasty (PTCA). METHODS AND RESULTS: In 102 patients with high-grade coronary stenosis admitted for elective PTCA, preprocedural platelet activation was characterized by flow cytometric measurement of the proteins CD62, CD63, and thrombospondin expressed on the platelet surface membrane. The prevalence of acute ischemic events during the 24 hours immediately after the procedure was then related to the pre-PTCA platelet activation status. Fifty-six patients were classified as "nonactivated," whereas 46 patients showed an increased percentage of activated platelets. Two patients developed acute occlusion (1.96%) and four patients high-grade restenosis (3.92%), as confirmed by second-look coronary angiography. All events occurred in patients classified as "activated" (six of 46, or 13%). None of these patients received beta-blocker medication, which was associated with lower expression of platelet membrane activation markers. In the nonactivated patient group, no clinical events were found (0 of 56, or 0%). This difference in prevalence is significant (p = 0.007). CONCLUSIONS: We conclude that analysis of platelet membrane activation markers may help to predict an increased risk of acute ischemic events after angioplasty.

Flow-cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets.

Occlusive vascular diseases are promoted by a "prethrombotic state" with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow-cytometric bio-assay and the related change in the actin status by using the DNase-I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F-actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein "actin" and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow-cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.

Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder.

Platelets from a patient with a severe lifelong bleeding tendency, which later spontaneously disappeared, lacked intact thrombospondin and glycoprotein (GP) Ia. Before disappearance of the bleeding disorder, results of coagulation studies and platelet aggregation in response to adenosine diphosphate (ADP), arachidonic acid, thrombin, A23187, epinephrine, and ristocetin were normal. In contrast, aggregation only occurred in the presence of collagen or wheat germ agglutinin at unusually high doses of these agonists. The platelets adhered normally to purified bovine and human type I collagen, and they did not spread in the presence of methylated type I collagen. No collagen-induced clot retraction was observed. Two-dimensional gel electrophoretic analyses of platelet proteins and immunologic studies showed that intact thrombospondin and GP Ia were absent. Aggregation in response to collagen could be restored by adding thrombospondin. Disappearance of the bleeding tendency occurred at the onset of menopause; subsequent analyses revealed that thrombospondin and GP Ia were present in platelets and that collagen-induced platelet aggregation was normal. These results suggest that both thrombospondin and GP Ia are essential in collagen-induced platelet aggregation. The spontaneous disappearance of the bleeding tendency may have been related to hormonal influences.