Thrombomodulin prolongs thrombin-induced extracellular signal-regulated kinase phosphorylation and nuclear retention in endothelial cells.

On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin.

Abnormal platelet cytoskeletal assembly in hemodialyzed patients results in deficient tyrosine phosphorylation signaling.

BACKGROUND: Uremic patients have a bleeding tendency associated with a platelet dysfunction. We evaluated the impact of a repeated hemodialysis procedure on primary hemostasis by analyzing different aspects of platelet activation in uremic patients. METHODS: Studies were performed in (1) eight patients with end-stage renal disease before the hemodialysis program was initiated and after initiating hemodialysis treatment, and in (2) eight patients on maintenance hemodialysis who were transferred to continuous ambulatory peritoneal dialysis. Studies included routine platelet aggregations and evaluation of platelet-subendothelium interactions under flow conditions. Contractile proteins and tyrosine phosphorylation associated with the cytoskeleton were analyzed, before and after thrombin activation of platelets, by electrophoresis after Triton X-100 extraction. RESULTS: No changes in the clinical parameters analyzed were observed among the different study groups. Aggregation and platelet adhesion only improved when patients were shifted from hemodialysis to continuous ambulatory peritoneal dialysis (P < 0.05 for both percentage of surface covered by platelets and aggregate formation). The association of cytoskeletal proteins in platelets from patients under hemodialysis treatment was statistically decreased with respect to the corresponding values in platelets from patients not subjected to dialysis (P < 0.01 for actin). However, after two months on peritoneal dialysis, these values increased to almost control values (P < 0.001 for actin, vs. hemodialysis). Similarly, translocation of tyrosine-phosphorylated proteins to the cytoskeletal fraction was impaired in platelets from hemodialyzed patients, and it recovered partially after the patients transferred to continuous ambulatory peritoneal dialysis. CONCLUSIONS: Our present data support the concept that repeated platelet stress during hemodialysis has a deleterious effect on the organization of platelet cytoskeleton, which seems to impair the translocation of signal transduction proteins within platelets compromising the platelet function in uremia.

Alterations in cytoskeletal organization and tyrosine phosphorylation in platelet concentrates prepared by the buffy coat method.

BACKGROUND: Numerous morphologic and biochemical changes occurring during platelet storage may result in the impairment of platelet function. STUDY DESIGN AND METHODS: The effect of preparation and storage conditions on platelet function was analyzed through evaluation of cytoskeletal organization and signaling mechanisms involved in the activation of platelets by thrombin. Samples of platelets prepared by the buffy coat method were obtained before and after the platelet concentrates were prepared during storage for 1, 3, and 5 days. Thrombin-induced aggregation was monitored, and changes in the organization of proteins in the cytoskeleton were analyzed by gel electrophoresis. For the analysis of tyrosine phosphorylation, proteins were transferred to nitrocellulose membranes and probed with a specific antibody. RESULTS: The aggregation and the cytoskeletal organization induced by thrombin activation were markedly impaired immediately after preparation of platelet concentrates, although they normalized after the first 24 hours of storage and decreased progressively after 3 days of storage. Results in tyrosine phosphorylation paralleled those obtained with cytoskeletal organization, except for samples obtained immediately after processing to obtain platelet concentrates. CONCLUSION: These data indirectly suggest that the stress induced by the preparation method has an activating effect on platelet function that may imply a delayed platelet response to further stimuli. This effect may result in a deficient redistribution of signaling molecules within platelets.

Thrombin facilitates primary platelet adhesion onto vascular surfaces in the absence of plasma adhesive proteins: studies under flow conditions.

BACKGROUND AND OBJECTIVE: The effect of local and circulating thrombin on platelet adhesion onto vascular surfaces was explored in the absence of plasma adhesive proteins using flow conditions. DESIGN AND METHODS: To study the local effects of thrombin, denuded rabbit aorta segments were incubated with thrombin concentrations of 0.001, 0.01 and 0.1 U/mL. To evaluate the effects of circulating thrombin, the same concentrations were added to perfusates consisting of washed platelets and washed red blood cells suspended in a human albumin solution (5%). In some experiments, purified von Willebrand's factor (vWF) (Haemate-P) was added to the perfusates (0. 8 U/mL of vWF, final concentration). A humanized chimeric antibody to the GPIIb-IIIa complex (Reopro) was used to determine the role of this glycoprotein on platelet adhesion under the conditions described. The effect of blocking GPIb was also assessed. Perfusions were carried out at 800 s(-1) for 10 min. The interaction of platelet with the vessel surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). Changes in the surface expression of the major platelet antigens were also analyzed by flow cytometry. RESULTS: Incubation of subendothelial surfaces with thrombin enhanced platelet deposition with respect to control levels (increases in SC of 64%, 79% and 86% with 0.001, 0.01 and 0.1 U/mL of thrombin, respectively). Low concentrations of thrombin (0.001 and 0.01 U/mL) incorporated in the perfusates resulted in a similar pro-adhesive effect (increases in SC of 64% and 71%, respectively) while the highest concentration (0.1 U/mL) failed to produce a pro-adhesive effect due to the augmented formation of platelet aggregates with subsequent thrombocytopenia (15+/-1 vs. 160+/-5x10(9) plt/L in the perfusates). Similar results were obtained when VWF was present in the perfusate. Reduction of platelet deposition by blockade of GPIIb-IIIa (to 5.3+/-0.7%) was partially restored by thrombin. Blockade of GPIb prevented platelets from adhering even when thrombin was present (%SC of 2.0+/-0.8%). No significant changes in the distribution of platelet membrane glycoproteins during perfusion experiments were detected. INTERPRETATION AND CONCLUSIONS: Our results suggest that thrombin facilitates primary platelet adhesion onto vascular surfaces even in the absence of plasma adhesive proteins. This effect seems to be mainly dependent on the GPIb/vWF axis.

Adherence of platelets under flow conditions results in specific phosphorylation of proteins at tyrosine residues.

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.

Platelet concentrates prepared and stored under currently optimal conditions: minor impact on platelet adhesive and cohesive functions after storage.

BACKGROUND: The effect on platelets of two standard methods of platelet concentrate (PC) preparation was studied by flow cytometry. The findings were correlated with those obtained in an experimental in vitro perfusion model. STUDY DESIGN AND METHODS: PCs were prepared from whole blood by the platelet-rich plasma (PRP) or buffy coat (BC) method and placed on a flatbed platelet agitator at 22 degrees C for up to 5 days. Platelet glycoproteins (GP)lbalpha, GPIIb/IIIa, and GPIV, p-selectin and lysosomal integral membrane protein, and the binding of von Willebrand factor, fibrinogen, fibronectin, and coagulation factor Va were measured with the corresponding specific conjugated antibodies. Perfusions were carried out in an annular chamber with citrated blood depleted of platelets and white cells by filtration, to which samples from PCs were added. RESULTS: PRP-PC production provoked intense platelet activation. In contrast, in BC-derived PCs, platelet activation was milder, and only a significant increase in bound fibrinogen was seen. After 1 day of storage, differences between the methods that had been observed immediately after separation had almost disappeared. During the remaining storage period, increases in activation-dependent antigens and in procoagulant activity were measured. Of the studied platelet GPs, only GPIIIb/ IIIa decreased by 25 percent in PRP-PCs. Differences in covered surface were not significant in perfusion studies performed on Day 0 and after 5 days of storage in PRP-PCs (26.8 +/- 6.9 vs. 20.5 +/- 5.8) or BC-PCs (23.8 +/- 11 vs. 24.8 +/- 10.2). CONCLUSION: Platelet activation occurred during the separation and storage of PCs prepared by both methods, and it was higher in PRP-PCs only in samples obtained immediately after preparation. Despite these changes, platelet adhesive and cohesive functions were similar in both types of PCs and remained basically unchanged after storage.

Cytoskeletal reorganization after preparation of platelet concentrates, using the buffy coat method, and during their storage.

BACKGROUND AND OBJECTIVE: The use of platelet transfusions has risen considerably in the last years. Changes occur in platelet biochemical and membrane properties during storage. We have analyzed the effect of platelet preparation and storage of platelet function through the evaluation of platelet cytoskeletal reorganization. METHODS: A blood sample was obtained from the donor and platelets were separated as standard platelet-rich plasma (PRP) (120 g, 20 min) (PRE sample). Aliquots were also collected immediately after preparation using buffy coat procedure of platelet concentrates (day 0) and after 1, 3 and 5 days of storage. Cytoskeleton composition in both low- and high-speed cytoskeletal fractions of detergent-lysed platelets was analyzed by gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Presence of each contractile protein was quantified by densitometry. RESULTS: The method used to prepare platelet concentrates induced actin polymerization (actin increased to 163.5 +/- 4.8%, mean +/- SEM, n = 8, p < 0.001, considering actin values in PRE sample as 100%) with a concurrent increase in the association of actin-binding protein (ABP), myosin and alpha-actinin to the low-speed cytoskeletal fraction. During the first 24 hours of storage, cytoskeletal assembly was partially reversed (134.8 +/- 2.6% of actin, p < 0.001) and actin polymerization increased gradually to 144.3 +/- 5.8% and 153.2 +/- 5.1% at days 3 and 5, respectively (p < 0.001 for both days). ABP, myosin and alpha-actinin showed similar tendencies to those referred for actin. Conversely, during platelet preparation and storage, the contractile proteins associated with the high-speed cytoskeletal fraction decreased, due to reorganization of the contractile proteins to the low speed fraction. INTERPRETATION AND CONCLUSIONS: The method used to prepare platelet concentrates (buffy coat procedure) induced cytoskeletal polymerization. This activating effect was partially reversed after 1 day of storage, although it increased progressively after 3 days of storage. The storage lesion may lead to defective cytoskeletal assembly in response to further stimulus. Analysis of cytoskeletal assembly is a sensitive method for detecting platelet activation caused by the concentrate preparation method and the storage conditions.