K. Vinod Vijayan

Enhanced Activation of Mitogen-activated Protein Kinase and Myosin Light Chain Kinase by the Pro33 Polymorphism of Integrin β3

Integrin β3 is polymorphic at residue 33 (Leu33 or Pro33), and the Pro33 variant exhibits increased outside-in signaling to focal adhesion kinase and greater actin reorganization. Because focal adhesion kinase activation and an intact cytoskeleton are critical links for integrin-mediated signaling to MAPK, we explored the role of integrin αIIbβ3 in this signaling using Chinese hamster ovary and human kidney 293 cell lines expressing either the Leu33 or Pro33 isoform of β3. Compared with Leu33 cells, Pro33 cells demonstrated substantially greater activation of ERK2 (but not MAPK family members JNK and p38) upon adhesion to immobilized fibrinogen (but not fibronectin) and upon integrin cross-linking. ERK2 activation was mediated through MAPK kinase and required phosphoinositide 3-kinase signaling and an intact actin cytoskeleton. Human platelets and Chinese hamster ovary cells expressing the Pro33 isoform showed enhanced activation of the ERK2 substrate myosin light chain kinase (MLCK) upon adhering to fibrinogen. Furthermore, compared with platelets and cells expressing the Leu33 isoform, the Pro33 variant showed greater α-granule release, clot retraction, and adhesion to fibrinogen under shear stress, and these functional differences were abolished by MLCK and MAPK kinase inhibition. Post-integrin occupancy signaling through MAPK and MLCK after αIIbβ3 cross-linking may explain in part the increased adhesive properties of the Pro33 variant of integrin β3.

PlA Polymorphism of Integrin β3 Differentially Modulates Cellular Migration on Extracellular Matrix Proteins

Objective—Cell migration is central to multiple physiological and pathologic processes and involves interactions between integrins on the cell surface and the extracellular matrix. The Leu33Pro (PlA) polymorphism of integrin &bgr;3 has been reported to be associated with a greater rate of restenosis after angioplasty, a process involving endothelial and smooth muscle cell migration. We have addressed the possibility that the Leu33Pro polymorphism could modify the migratory behavior of Chinese hamster ovary (CHO) cells expressing the &bgr;3-containing integrin complexes. Methods and Results—Haptotactic migratory responses of CHO &agr;IIb&bgr;3 cells to fibronectin and vitronectin were not statistically different between the Leu33 and Pro33 cells. However, CHO cells with the Pro33 (PlA2) polymorphism had an enhanced haptotactic migratory response to fibrinogen and von Willebrand Factor. This enhanced migration (1) could be blocked by the &agr;IIb&bgr;3-complex–specific neutralizing mAb 10E5, by 7E3, a neutralizing mAb specific for the &bgr;3 integrin, and by the &agr;IIb&bgr;3-blocking peptide Integrilin; (2) was not observed with a CHO cell line expressing an activating &bgr;3 Cys435 to Ala mutation; and (3) was attributable to increased activity of mitogen-activated protein kinase and cyclooxygenase. CHO cell lines expressing the Pro33 isoform of &agr;v&bgr;3 had an enhanced haptotactic migratory response to vitronectin and osteopontin but not fibrinogen. Conclusions—The Leu33Pro polymorphism alters the migratory behavior of cells on extracellular matrix substrates, and the &agr; subunit influences the substrate specificity of this genetic effect.

Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ibα and Platelet Activation under Shear Stress

This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ibα (GPIbα). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIbα without ristocetin with an apparent KD of 85 ± 34 nm, comparable with that of WT (36 ± 12 nm) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s−1 shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s−1, platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIbα and αIIbβ3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.

Decline in platelet microparticles contributes to reduced hemostatic potential of stored plasma

INTRODUCTION In an effort to administer life-saving transfusions quickly, some trauma centers maintain thawed plasma (TP). According to AABB, TP is approved for transfusion for up to five days when stored at 1-6° C. However, the alterations in microparticles (MP) contained in the plasma, which are an integral component of plasmas hemostatic capacity, are not well characterized. We report on MP changes in TP between its initial thaw (FFP-0) and five days (FFP-5) of storage. MATERIALS AND METHODS FFP units (n=30) were thawed at 37° C and kept refrigerated for five days. Phenotypes of residual cells, which include platelets, erythrocytes, leukocytes, monocytes, endothelial cells, and MP counterparts of each cell type, were analyzed by flow cytometry. Functional assays were used for MP procoagulant activity, plasma thrombin generation, and clotting properties (thromboelastography). RESULTS In FFP-0 the majority (94%) of residual cells were platelets, along with significant levels of platelet MPs (4408 × 10(3)/L). FFP-5 showed a decline in MP count by 50% (p<0.0001), and procoagulant activity by 29% (p<0.0001). FFP-5 exhibited only 54% (p<0.0001) of the potential for thrombin generation as FFP-0, while thromboelastography indicated a slower clotting response (p<0.0001) and a longer delay in reaching maximum clot (p<0.01). Removal of MP by filtration resulted in reduced thrombin generation, while the MP replacement restored it. CONCLUSIONS Decline in MP with storage contributes to FFP-5s reduced ability to provide the hemostatic potential exhibited by FFP-0, suggesting the presence of platelet MPs in freshly TP may be beneficial and protective in the initial treatment of hemorrhage.

Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a Gq blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y1 and Gq knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin αIIbβ3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a αIIbβ3 receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, αIIbβ3 failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y1 receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by αIIbβ3 integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

Association between cell-derived microparticles and adverse events in patients with nonpulsatile left ventricular assist devices

BACKGROUND Continuous-flow left ventricular assist devices (LVADs) expose blood cells to high shear stress, potentially resulting in the production of microparticles that express phosphatidylserine (PS+) and promote coagulation and inflammation. In this prospective study, we attempted to determine whether PS+ microparticle levels correlate with clinical outcomes in LVAD-supported patients. METHODS We enrolled 20 patients undergoing implantation of the HeartMate II LVAD (Thoratec Corp, Pleasanton, CA) and 10 healthy controls who provided reference values for the microparticle assays. Plasma was collected before LVAD implantation, at discharge, at the 3-month follow-up, and when an adverse clinical event occurred. We quantified PS+ microparticles in the plasma using flow cytometry. RESULTS During the study period, 8 patients developed adverse clinical events: ventricular tachycardia storm in 1, non-ST-elevation myocardial infarction in 2, arterial thrombosis in 2, gastrointestinal bleeding in 2, and stroke in 3. Levels of PS+ microparticles were higher in patients at baseline than in healthy controls (2.11% ± 1.26% vs 0.69% ± 0.46%, p = 0.007). After LVAD implantation, patient PS+ microparticle levels increased to 2.39% ± 1.22% at discharge and then leveled to 1.97% ± 1.25% at the 3-month follow-up. Importantly, levels of PS+ microparticles were significantly higher in patients who developed an adverse event than in patients with no events (3.82% ± 1.17% vs 1.57% ± 0.59%, p < 0.001), even though the 2 patient groups did not markedly differ in other clinical and hematologic parameters. CONCLUSIONS Our results suggest that an elevation of PS+ microparticle levels may be associated with adverse clinical events. Thus, measuring PS+ microparticle levels in LVAD-supported patients may help identify patients at increased risk for adverse events.

Signal Transducer and Activator of Transcription 3 (STAT3) Regulates Collagen-Induced Platelet Aggregation Independently of Its Transcription Factor Activity

Background— Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. Methods and Results— The signal transducer and activator of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk (spleen tyrosine kinase) and the substrate PLC&ggr;2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLC&ggr;2 was detected in HEK293 cells transfected with cDNAs for Syk and PLC&ggr;2 and stimulated with interleukin-6. Pharmacological inhibition of STAT3 blocked ≈50% of collagen- and a collagen-related peptide–induced but not thrombin receptor–activating peptide– or ADP-induced aggregation and ≈80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet-specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine interleukin-6 enhanced the collagen-induced STAT3 activation in human platelets. Conclusions— These data demonstrate a nontranscriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for the platelet hyperactivity found in conditions of inflammation.Background —Platelet hyperactivity induced by inflammation is a known risk factor for atherosclerosis and thrombosis, but its underlying mechanisms remain poorly understood. Methods and Results —The signal transducers and activators of transcription 3 (STAT3) was activated in collagen-stimulated platelets. Activated STAT3 served as a protein scaffold to facilitate the catalytic interaction between the kinase Syk and the substrate PLCγ2 to enhance collagen-induced calcium mobilization and platelet activation. The same interaction of STAT3 with Syk and PLCγ2 was also detected in HEK293 cells transfected with cDNAs for Syk and PLCγ2, and stimulated with interleukin-6 (IL-6). Pharmacological inhibition of STAT3 blocked ~50% of collagen- and a collagen-related peptide-, but not TRAP- or ADP-induced aggregation and ~80% of thrombus formation of human platelets on a collagen matrix. This in vitro phenotype was reproduced in mice infused with STAT3 inhibitors and mice with platelet specific STAT3 deficiency. By forming a complex with its soluble receptor, the proinflammatory cytokine IL-6 enhanced the collagen-induced STAT3 activation in human platelets. Conclusions —These data demonstrate a non-transcriptional activity of STAT3 that facilitates a crosstalk between proinflammatory cytokine and hemostasis/thrombosis signals in platelets. This crosstalk may be responsible for platelet hyperactivity found in conditions of inflammation.

Shear stress augments the enhanced adhesive phenotype of cells expressing the Pro33 isoform of integrin β3

Adhesion of platelets to the exposed extracellular matrix proteins at sites of vascular injury is partly regulated by the local fluid shear stress. Because the Leu33Pro (PlA) polymorphism of integrin β3 confers only a modest increase in adhesion under static conditions, we used CHO and 293 cells expressing the Leu33 or Pro33 isoform of β3 in flow chamber experiments to test whether shear forces would alter the PlA adhesive phenotype. We found that shear force augmented the Pro33‐mediated enhanced adhesion to fibrinogen. This Pro33‐dependent enhancement was aspirin‐sensitive and was also observed on immobilized von Willebrand factor and cryoprecipitate, but not fibronectin. Thus, shear stress enhances the adhesive phenotype of the Pro33 cells to multiple physiologic substrates.

Platelets and Their Interactions with Other Immune Cells.

Platelets are anucleate blood cells, long known to be critically involved in hemostasis and thrombosis. In addition to their role in blood clots, increasing evidence reveals significant roles for platelets in inflammation and immunity. However, the notion that platelets represent immune cells is not broadly recognized in the field of Physiology. This article reviews the role of platelets in inflammation and immune responses, and highlights their interactions with other immune cells, including examples of major functional consequences of these interactions.

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Significance Platelets are known to be both numerically and functionally altered in some patients with cancer. However, structural differences in the platelets from these patients have not been studied. Here we use electron cryotomography to reveal that, compared with control donors, the microtubule system and the mitochondria of platelets from patients diagnosed with ovarian cancer are significantly different. This finding suggests the potential of electron cryotomography as a technology to detect structural biomarkers of diseases affecting platelets. Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.