Heritability of platelet function in families with premature coronary artery disease.

BACKGROUND: Variations in platelet function among individuals may be related to differences in platelet-related genes. The major goal of our study was to estimate the contribution of inheritance to the variability in platelet function in unaffected individuals from white and African American families with premature coronary artery disease. METHODS: Platelet reactivity, in the absence of antiplatelet agents, was assessed by in vitro aggregation and the platelet function analyzer closure time. Heritability was estimated using a variance components model. RESULTS: Both white (n = 687) and African American (n = 321) subjects exhibited moderate to strong heritability (h(2)) for epinephrine- and adenosine diphosphate-induced aggregation (0.36-0.42 for white and >0.71 for African American subjects), but heritability for collagen-induced platelet aggregation in platelet-rich plasma was prominent only in African American subjects. Platelet lag phase after collagen stimulation was heritable in both groups (0.47-0.50). A limited genotype analysis demonstrated that the C825T polymorphism of GNB3 was associated with the platelet aggregation response to 2 muM epinephrine, but the effect differed by race. CONCLUSIONS: Considering the few and modest genetic effects reported to affect platelet function, our findings suggest the likely existence of undiscovered important genes that modify platelet reactivity, some of which affect multiple aspects of platelet biology.

Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin.

BACKGROUND: Platelet--leukocyte conjugates have been observed in patients with unstable coronary syndromes and after cardiopulmonary bypass. In vitro, the binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL1) mediates conjugate formation; however, the hemostatic implications of these cell--cell interactions are unknown. The aims of this study were to determine the ability of leukocytes to modulate platelet agonist--induced aggregation and secretion in the blood milieu, and to investigate the role of P-selectin and PSGL-1 in mediating these responses. METHODS: Blood was drawn from healthy volunteers for in vitro analysis of platelet agonist--induced aggregation, secretion (adenosine triphosphate, beta-thromboglobulin, and thromboxane), and platelet-leukocyte conjugate formation. Experiments were performed on live cells in whole blood or plasma to simulate physiologic conditions. Whole-blood impedance and optical aggregometry, flow cytometry, and enzyme-linked immunosorbent assays were performed in the presence and absence of blocking antibodies to P-selectin and PSGL1. The platelet-specific agonists, thrombin receptor activating peptide and adenosine diphosphate, were used to elicit platelet activation responses. RESULTS: Inhibition of platelet--leukocyte adherence by P- selectin and PSGL1 antibodies decreased agonist--induced aggregation in whole blood. The presence of leukocytes in platelet-rich plasma increased aggregation, and this increase was attenuated by P-selectin blocking antibodies. Data from flow cytometry confirmed that platelet-leukocyte conjugate formation contributed to aggregation responses. Blocking antibodies reduced platelet agonist--induced thromboxane release but had no impact on adenosine triphosphate and beta-thomboglobulin secretion. CONCLUSIONS: Leukocytes can enhance platelet agonist--induced aggregation and thromboxane release in whole blood and platelet-rich plasma under shear conditions in vitro. Interaction of platelet P-selectin with leukocyte PSGL1 contributes substantially to these effects.

Human megakaryocytes and platelets contain the estrogen receptor beta and androgen receptor (AR): testosterone regulates AR expression.

Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34(+) stem cells contained RNA for ER beta and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER beta and AR transcripts. No ER alpha or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER beta protein was present in glycoprotein (GP) IIb(+) megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb(+) and GPIIb(-) cells derived from CD34(+) cells and in HEL cells. Western immunoblotting confirmed the presence of ER beta and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases.

Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression.

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.

In vitro hypothermia enhances platelet GPIIb-IIIa activation and P-selectin expression.

BACKGROUND: A clinical bleeding diathesis is associated with hypothermia. Inhibition of platelet reactivity is the purported cause of this coagulopathy despite inconsistent evidence to support this hypothesis. To clarify the effect of temperature on intrinsic platelet function, platelet GPllb-IIIa activation and P-selectin expression were assessed under normothermic and hypothermic conditions in vitro. METHODS: Blood was obtained by venipuncture from healthy volunteers. Platelet activation was assessed by aggregometry and by cytometric analysis of platelet binding of fibrinogen, PAC-1, and P-selectin antibodies. Measurements were made at normothermia (37 degrees C), moderate hypothermia (33 degrees C), and profound hypothermia (22 degrees C) after stimulating samples with adenosine diphosphate (ADP), collagen, or thrombin receptor activating peptide. RESULTS: Agonist-induced platelet aggregation and fibrinogen binding were significantly greater at 22 degrees C and 33 degrees C than at 37 degrees C. Platelet fibrinogen binding values to 20 micro M ADP were 23,400, 14,300, and 9,700 molecules/platelet at 22 degrees C, 33 degrees C, and 37 degrees C, respectively. The aggregation responses of platelets that were cooled and rewarmed were indistinguishable from those of platelets maintained at 37 degrees C throughout the study. Platelet binding of PAC-1 and P-selectin antibodies was greater under hypothermic conditions. CONCLUSIONS: Aggregation, fibrinogen binding, PAC-1 binding, and P-selectin antibody binding studies showed that platelet GPIIb-IIIa activation and alpha-granule release were enhanced at hypothermic temperatures. Thus hypothermia appears to increase the ability of platelets to respond to activating stimuli. The coagulopathy associated with hypothermia is not likely to be the result of an intrinsic defect in platelet function.

Neuroendocrine stress hormones do not recreate the postoperative hypercoagulable state.

UNLABELLED: Surgery causes changes in hemostasis, leading to a hypercoagulable state that has been linked to both arterial and venous thrombotic complications. The etiology of this state is unknown, but many investigators have hypothesized that perioperative neuroendocrine changes are responsible. We have previously demonstrated minimal increases in hemostatic function with a stress hormone infusion. This study was undertaken to further examine the relationship between neuroendocrine hormones and hemostatic function. Seventeen healthy volunteers were administered a stress hormone cocktail i.v. (epinephrine, cortisol, glucagon, angiotensin II, and vasopressin) for 24 h in a blind, placebo-controlled, cross-over design in our clinical research center. Venous blood samples were obtained for measurement of hemostatic function before the infusion and at 2, 8, and 24 h. There were no demonstrable increases in any measure of hypercoagulability. Alternatively, there was an increase in tissue plasminogen activator and protein C activity. These changes are consistent with an inhibition of coagulation and improved fibrinolysis. These data suggest that this combination of neuroendocrine hormones is not responsible for the postoperative hypercoagulable state. IMPLICATIONS: Infusion of five stress hormones (epinephrine, cortisol, glucagon, vasopressin, and angiotensin II) to normal volunteers does not cause increases in procoagulant proteins and platelet reactivity or decreases in fibrinolytic proteins. Alternatively, these five hormones caused increased levels of fibrinolytic proteins (tissue plasminogen activator) and endogenous anticoagulants (protein C antigen and activity).

Gender differences in platelet GPIIb-IIIa activation.

Gender differences in the development of thrombotic diseases have been described in numerous clinical settings. Enhanced platelet reactivity in both sexes is associated with the development of vascular thromboses. Because activation of platelet GPIIb-IIIa receptors is a central event in thrombus formation, we examined GPIIb-IIIa function in normal male and female volunteers. Using flow cytometry, we quantitated gender differences in the number of binding sites for FITC-labeled fibrinogen (FITC-FGN) and FITC-labeled PAC-1 antibody (FITC-PAC-1). Washed platelets were incubated with either FITC-FGN or FITC-PAC-1 and activated with either ADP or thrombin receptor activating peptide (TRAP) prior to cytometric acquisition of data. The dissociation constant for FITC-FGN was the same in both sexes (approx. 1.6 x 10(-7)M), however, the number of GPIIb-IIIa receptors per platelet capable of binding fibrinogen was significantly greater in women than men in response to 2 microM ADP (16,319 +/- 1871 vs 9669 +/- 1994, p = 0.02), 20 microM ADP (39,951 +/- 4711 vs 25,948 +/- 4953, p = 0.05) and 50 microM TRAP (39,236 +/- 3965 vs 21,848 +/- 4159, p = 0.007). Similarly, the number of GPIIb-IIIa receptors capable of binding PAC-1 in response to ADP and TRAP was 50% to 80% greater in women than men. Binding experiments using specific anti-GPIIb-IIIa monoclonal antibodies (P2 and 10E5), as well as quantitative Western blotting experiments, showed no gender difference in the total number of GPIIb-IIIa molecules expressed. Analysis of data from female subgroups demonstrated an association of GPIIb-IIIa reactivity with menstrual phase. We conclude that GPIIb-IIIa receptors on platelets from premenopausal women are more "activatable" than those on platelets from young men. Variations in the serum concentrations of estrogens and/or progestins may modulate GPIIb-IIIa function.

Effects of storage time on quantitative and qualitative platelet function after transfusion.

BACKGROUND: Platelet transfusions are being used increasingly in patients with thrombocytopenia to improve hemostatic function before surgery and invasive procedures. However, there are limited data on the immediate quantitative and qualitative platelet response after transfusion. Some authors have suggested that transfused platelets require time in vivo to regain maximal function, which is dependent on the duration of platelet storage. Therefore, the timing of surgery and invasive procedures with optimal platelet function may not be occurring. METHODS: Twenty-five patients with thrombocytopenia from ablation chemotherapy and total body irradiation (before bone marrow transplantation), were randomized to receive either 1-day (fresh) or 4-day stored platelets. No patient had infection, organ system dysfunction, or previous platelet transfusion. Single-donor platelets were transfused (1 unit/10 kg body weight) over 60 min. Whole blood from an indwelling central venous catheter was obtained before, immediately after, and 1, 2, and 24 h after transfusion. Platelet number was measured on a Coulter counter and platelet reactivity was measured on a Coulter counter and platelet reactivity was measured using agonist-induced whole blood impedance aggregometry (ohms) and dense granule release (adenosine triphosphate [ATP]). RESULTS: Platelet number increased similarly (21,000 +/- 2,000/mm3 to 76,000 +/- 7,000/MM3 AND 20,000 +/- 1,000/MM3 TO 65,000 +/- 4,000/MM3) after transfusion in the 1- and 4-day stored platelets, respectively. These levels were maintained for 2 h after transfusion in both groups and then decreased similarly (26% and 27%) at 24 h. Agonist-induced platelet aggregation increased immediately after transfusion to 5 micrograms/ml collagen (0.7 +/- 0.4 to 11.4 +/- 1.0 ohms and 0.1 +/- 0.1 to 5.2 +/- 1.0 ohms), 10 micrograms/ml collagen, (1.5 +/- 0.7 to 18.0 +/- 1.9 ohms and 0.6 +/- 0.4 to 10.0 +/- 1.6 ohms) and ristocetin (0.7 +/- 0.4 to 10.1 +/- 1.7 and 0.1 +/- 0.7 to 6.2 +/- 1.0 ohms), in 1- and 4-day, stored platelets, respectively and persisted unchanged in both groups for 2 h. Fresh platelets were hyperaggregable compared to 4-day stored platelets for collagen-induced (5 micrograms/ml and 10 micrograms/ml) aggregation. Agonist-induced platelet dense granule release (ATP) increased immediately after transfusion to 5 micrograms/ml collagen (42 +/- 18 to 410 +/- 49 picomoles ATP and 20 +/- 7 to 186 +/- 22 picomoles ATP), 10 micrograms/ml collagen (60 +/- 22 to 449 +/- 53 picomoles ATP and 44 +/- 13 to 219 +/- 25 picomoles ATP in 1- and 4-day platelets, respectively. Ristocetin-induced ATP release increased immediately after transfusion of fresh platelets only (0 +/- 0 to 69 +/- 17) and remained unchanged for 2 h. Fresh platelets also demonstrated greater dense granule release to collagen (5 micrograms and 10 micrograms/ml) and ristocetin than 4-day stored platelets. CONCLUSIONS: In patients with chemotherapy-induced thrombocytopenia, platelet transfusion causes an immediate increase in number and function, which is independent of storage time. This quantitative and qualitative increase persists unchanged for 2 h after transfusion, suggesting that there is no acute "warm-up-time" necessary for transfused platelets to regain maximal function. Fresh platelets demonstrate increased aggregation and dense granule release compared to 4-day stored platelets and may impart improved hemostatic function in vivo.

Surface recruitment but not activation of integrin alpha IIb beta 3 (GPIIb-IIIa) requires a functional actin cytoskeleton.

Binding of integrin alpha IIb beta 3 (glycoprotein [GP] IIb-IIIa) to soluble fibrinogen requires that the receptor undergo a conformational change (receptor activation), which occurs rapidly in agonist-stimulated platelets. Agonist stimulation of platelets also results in alpha IIb beta 3 recruitment from intracellular membranes (alpha-granules and open canalicular system) to the platelet surface. Once activated and accessible, the receptor can engage, a process that corresponds to the binding of the receptor to its soluble fibrinogen ligand, leading to intracellular signaling reactions and centripetal migration of bound receptor molecules. Because these processes occur concurrently with a marked reorganization of the actin cytoskeleton, we investigated the role of actin in fibrinogen receptor activation and surface recruitment. We used a flow cytometric assay to directly quantitate the binding of alpha IIb beta 3 to fluorescently labeled fibrinogen on the platelet surface. Cytochalasin D, which inhibits elongation of actin filaments, was used to prevent the actin response to platelet agonists. Despite its ability to inhibit the actin response and alpha IIb beta 3 binding to the actin cytoskeleton, cytochalasin D did not alter the agonist-induced intramolecular changes resulting in increased affinity of alpha IIb beta 3 for soluble fibrinogen and therefore did not inhibit ADP-induced aggregation. Thus, disruption of the actin network with cytochalasin D had no effect on the dissociation constant of the complex between activated alpha IIb beta 3 and fibrinogen (Kd = 0.26 to 0.28 mumol/L). However, cytochalasin D suppressed the recruitment of cryptic alpha IIb beta 3 molecules to the platelet surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemostatic effects of stress hormone infusion.

BACKGROUND: Surgery causes changes in hemostasis, leading to a hypercoagulable state. This postoperative increase in hemostatic function is attenuated in patients receiving regional anesthesia compared with those receiving general anesthesia. Regional anesthesia also decreases the neuroendocrine response to surgery compared with general anesthesia, and this effect is hypothesized to be responsible for the differences in hemostatis. To test the hypothesis that neuroendocrine hormones cause changes in hemostasis, we infused stress hormones into normal volunteers and measured hemostatic function. METHODS: After drug screening, 12 normal volunteers were studied. On two admissions, volunteers randomly received either stress hormone (epinephrine, cortisol, or glucagon) or placebo infusion for 24 h. During infusion, patients remained at bed rest and received controlled meals. Blood was obtained from indwelling venous catheters before infusion and 2, 8, and 24 h after the start of infusion. Blood was analyzed for neuroendocrine hormone concentrations, glucose, complete blood count, coagulation proteins, platelet reactivity, and activity of the fibrinolytic system. RESULTS: In the stress hormone group, concentrations of epinephrine, norepinephrine, cortisol, glucagon, and insulin were increased during the infusion period compared with those in the placebo group. Glucose concentrations and white blood cell counts were increased in the stress hormone group compared with those in the placebo group. Circulating fibrinogen concentrations increased 30% and ex vivo collagen-induced platelet reactivity increased 123% (aggregation) and 103% (dense granule release) in the stress hormone infusion group, whereas there was no change in the placebo group. Fibrinolytic proteins were similar in both groups, demonstrating a decrease in plasminogen activator inhibitor-1 activity at 8 and 24 h (196% in the hormone group vs. 199% in the placebo group). CONCLUSIONS: Infusion of stress hormones to concentrations found during surgery is safely tolerated and causes metabolic changes observed with surgery. Stress hormone infusion increases ex vivo platelet reactivity and fibrinogen concentrations that resemble changes seen postoperatively but does not recreate the postoperative decrease in fibrinolytic activity. Differences in neuroendocrine response between types of anesthesia may explain some postoperative changes in platelet function and acute phase reactivity, but additional uncharacterized factors are responsible for the differences in fibrinolysis.

The effects of bedrest on circadian changes in hemostasis.

Venous stasis occurs when people are at bedrest, because of altered venous flow characteristics. This is commonly believed to be one etiology behind the development of deep venous thrombosis (DVT). The hemostatic effects of bedrest and their possible role in DVT development have not been fully examined. We hypothesized that bedrest would lead to increases in hemostatic function and that these increases could be important in the development of DVT. Twelve non-smoking volunteers were studied during supine positioning for 36 hours. Platelet reactivity and plasma concentrations of fibrinogen, alpha 2-antiplasmin, plasminogen, thromboxane beta 2, plasminogen activator inhibitor-1, tissue plasminogen activator and neuroendocrine hormones (cortisol, epinephrine and norepinephrine) were measured at 8:00 a.m., 10:00 a.m., 4:00 p.m. and 8:00 a.m. Cortisol demonstrated an early morning increase while catecholamines were unchanged throughout. Fibrinogen, alpha 2-antiplasmin, plasminogen and platelet reactivity were no different at any time point. Fibrinolytic proteins changed over time, manifested by decreased PAI-1 antigen and activity levels at 24 h. Based upon the parameters measured, bedrest causes no increase in hemostatic function. In fact, bedrest causes the potential for enhanced fibrinolysis, that differs from that previously reported for normal activity over 24 h. This may represent a protective mechanism to counter the effects of stasis from bedrest.

Quantitation of soluble fibrinogen binding to platelets by fluorescence-activated flow cytometry.

Soluble fibrinogen binding to agonist-stimulated blood platelets is the essential physiologic function of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor. We describe a method of quantifying this receptor-ligand interaction by using flow cytometry to detect the binding of fluorescein-labeled fibrinogen to activated platelets. Fibrinogen conjugated with fluorescein isothiocyanate (FITC-FGN) was structurally and functionally indistinguishable from native fibrinogen when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thrombin clottability, and receptor affinity studies. Platelet samples, at a concentration of 2 x 10(7) ml, were incubated with FITC-FGN and activated with adenosine diphosphate (ADP) before cytometric acquisition of fluorescence and scatter data. ADP-induced binding of soluble FITC-FGN to platelet GPIIb-IIIa was specific, time dependent, and saturable. Cytometric analysis of FITC calibration beads allowed generation of standard curves relating bead fluorescence intensity to the number of fluorescein equivalents per bead. With this information, platelet fluorescence intensity was converted into the number of FITC-FGN molecules bound per platelet. Such quantitative analysis of fibrinogen binding yielded a dissociation constant of 2.48 +/- 0.5 x 10(-7) mol/L and a maximum fibrinogen binding capacity of 42, 124 +/- 5, 628 molecules per platelet (mean +/- SEM), which are comparable to published results with radioligand assays. The simplicity, sensitivity, and quantifiability of this method may make it a useful technique for basic and clinical research involving GPIIb-IIIa function.

Perioperative platelet reactivity and the effects of clonidine.

BACKGROUND: Increased postoperative platelet reactivity may contribute to arterial thrombotic complications following surgery. alpha 2 Agonists, which are being used increasingly to blunt the stress response of surgery, increase platelet aggregation in vitro. We compared perioperative changes in platelet reactivity in 21 patients receiving either clonidine or placebo. METHODS: Patients undergoing major abdominal surgery were randomized to receive oral and transdermal clonidine (n = 11) or placebo (n = 10). All patients received similar perioperative management, including preoperative sedation, general anesthesia without neuraxial opioids, or local anesthetics and postoperative patient-controlled intravenous morphine. Blood was obtained for measurement of clonidine level, fibrinogen concentration, platelet count, and platelet reactivity (impedance aggregometry and dense granule release) before induction and 24, 48, and 72 h postoperatively. RESULTS: Thirteen of the 21 patients had biopsy-proven cancer at surgery, 5 of 11 received clonidine and 8 of 10 received placebo (NS). Clonidine levels were therapeutic (1-2 ng/ml) throughout the study period. Clonidine administration had no effect on platelet count or platelet reactivity. Therefore, the groups were combined for further analysis. In this group (n = 21), compared to preoperative values, fibrinogen levels rose maximally (36%) at 72 h postoperatively and platelet counts decreased 22% at 48 h. Platelet reactivity (aggregation and degranulation) to collagen, adenosine diphosphate, arachidonic acid, and ristocetin, increased at 24, 48, and 72 h postoperatively. Thrombin-induced (supramaximal stimulus) dense granule release did not change from preoperative values. CONCLUSIONS: These data indicate that major abdominal surgery causes increased platelet reactivity postoperatively but does not effect maximal degranulation. This increased platelet reactivity occurs within 48 h of surgery, coinciding with the peak incidence of postoperative arterial thrombotic complications. Clonidine administration has no effect on surgically induced changes in platelet reactivity. In this surgical patient population, the use of clonidine should not increase the risk of platelet-induced perioperative arterial thrombosis.