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Objective: To test the hypothesis that statins will acutely inhibit platelet thrombus formation, intravenous lovastatin was assessed in our well-characterized porcine carotid injury model., Methods and Results: The first carotid artery was crush-injured and harvested after 30 minutes. Pigs then received intravenous lovastatin (100 microg/kg bolus+100 microg/kg/h infusion, n=6) or saline (n=11) before injury of the second carotid artery. Thrombus size was quantified by scintillation detection of autologous (111)In-platelets. Sequential carotid injury produced a thrombus more than 50% greater in volume in the second (3149+/-2053 x 10(6)/cm(2)) relative to the first injured artery (2081+/-1552 x 10(6)/cm(2); P=0.04) in control pigs. This augmentation was inhibited by intravenous lovastatin which acutely reduced platelet deposition (944+/-246 x 10(6)/cm(2)) relative to saline control (P=0.02). Flow chamber closure times increased on average by 2.45-fold in response to whole blood lovastatin incubation. Lovastatin (P<0.05) and simvastatin (P<0.05) reduced platelet dense granule secretion in vitro., Conclusions: Sequential arterial injury augments the thrombotic response suggesting that the propensity for arterial thrombosis is at least partially acquired. This thrombotic augmentation can be acutely attenuated by intravenous lovastatin which may result from a pleiotropic impact on platelet function. These results appear to be a class effect of 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors.

Background: Although exogenous estrogenic therapies increase the risk of thrombosis, the effects of estrogen on formed elements of blood are uncertain., Objective: This article examines the genomic and nongenomic actions of estrogen on platelet phenotype that may contribute to increased thrombotic risk., Methods: To determine aggregation, secretion, protein expression, and thrombin generation, platelets were collected from experimental animals of varying hormonal status and from women enrolled in the Kronos Early Estrogen Prevention Study., Results: Estrogen receptor beta predominates in circulating platelets. Estrogenic treatment in ovariectomized animals decreased platelet aggregation and adenosine triphosphate (ATP) secretion. However, acute exposure to 17beta-estradiol did not reverse decreases in platelet ATP secretion invoked by lipopolysaccharide. Thrombin generation was positively correlated to the number of circulating microvesicles expressing phosphatidylserine., Conclusion: Assessing the effect of estrogen treatments on blood platelets may lead to new ways of identifying women at risk for adverse thrombotic events with such therapies.

1. Adverse thrombotic cardiovascular events increase in women coincident with the onset of menopause. 2. Age past menopause may be an important variable in defining the benefit/risk of hormone treatments. 3. Few studies have examined hormonal status as a variable of ageing using a polygenomic approach of both humoral and cellular components of the coagulation system. 4. Longitudinal studies of a global set of platelet functions that define procoagulant activity (i.e. adhesion, aggregation, secretion and thrombin production) in individuals with documented hormonal status are needed to better understand how hormonal changes associated with ageing impact thrombotic risk.

Gram-negative bacteria release LPS, which activates Toll-like-receptor-4 (TLR4) in the host, initiating an inflammatory response to infection. Infection increases risk for thrombosis. Platelets contribute to defense from infection and to thrombosis. Experiments were designed to determine whether LPS, through TLR4 signaling, affects platelet phenotype. Platelet responses in wild-type (WT) mice and mice that lack the TLR4 gene (dTLR4) were compared following a single nonlethal injection of LPS (0.2 mg/kg iv). Compared with WT mice, mice without TLR4 had fewer circulating platelets with lower RNA content and were less responsive to thrombin-activated expression of P-selectin but were equally sensitive to aggregation or ATP secretion. One week following the LPS injection, the time it takes for the circulating platelet pool to turnover, the number of circulating platelets, thrombin-induced expression of P-selectin, and collagen-activated aggregation were increased comparably in both groups of mice. Therefore, the change of the platelet pool to an activated phenotype 1 wk after a single exposure to LPS appears to arise from a process that is independent of TLR4. The persistence of the effect 1 wk after the injection suggests that the changes reflect an action of LPS on megakaryocytes and their platelet progeny rather than on circulating platelets, which would have been cleared.

Post-menopausal hormone therapy increases the risk for venous thrombosis, and possibly myocardial infarction (MI) and ischemic stroke. However, most women using hormone therapy do not suffer thrombosis, and to date our ability to identify women at risk is limited. Thrombosis, arterial or venous, has 2 requisites: a vascular anomaly and a response of the hemostasis system to the anomaly. Consequently, experimental approaches to understand the pathophysiology of thrombosis require definition of vascular anatomy and function as well as characteristics of the blood within the context of genetic background, lifestyle choices and environmental exposures, which influence gene expression. Defining interactions among factors that affect individual propensity to thrombosis will allow physicians to better identify at-risk individuals, for example a woman contemplating estrogen therapy for symptoms of menopause, and prevent adverse thrombotic events.

Background/objective: Thromboembolism secondary to atrial fibrillation accounts for approximately one-fourth of all strokes. Although considerable resources have been targeted to pharmacologic prophylaxis, neither the cellular nor the biochemical composition of atrial thrombi is known. Quantitative immunohistochemistry was undertaken to define the composition of atrial thrombi and to explore morphological differences between atrial appendage thrombi and those that embolize., Patients/methods: Serial sections of thrombi obtained during valve replacement surgery or embolectomy from 22 patients with atrial fibrillation were stained with antibodies against fibrin, integrin beta3, or tissue factor and analyzed with NIH-image., Results: Thrombi showed distinct regions staining for either fibrin or platelets and on average, the fibrin-rich regions predominated (P < 0.0001). The platelet content of embolized thrombi was nearly twice that of atrial thrombi (P = 0.02). Non-staining amorphous material comprised nearly half of atrial thrombi in situ, but was rare in embolized thrombi (P < 0.001). Tissue factor colocalized to areas rich in platelets and granulocytes., Conclusions: The abundance of fibrin relative to platelets underscores the enhanced efficacy of warfarin prophylaxis in clinical trials. The finding of tissue factor localized to platelet-leukocyte clusters suggests its blood-borne origin. Compositional differences between in situ and embolized thrombi suggest directions for investigating propensity for embolization.

Objective: Occurrence of arterial thrombosis secondary to vascular disease in an individual is not easily predicted. After establishing that this poor predictability arises at least in part from an intrinsic thrombosis propensity of the individual, we sought to determine whether the propensity for arterial thrombosis is governed by blood or arterial wall factors., Methods and Results: To evaluate the variability arising from the blood, autologous 111In-labeled platelet deposition was measured after high-shear perfusion of compressed aortic strips, prepared from a single pig, with heparinized blood from 25 pigs. To evaluate the variability arising from the vessel wall, aortic strips from 8 pigs were superfused with blood from a single animal. Blood samples from 25 animals superfused over aortic substrate from a single source yielded a 24-fold range of platelet deposition. In contrast, when aortic substrates from 8 different animals were superfused with blood from a single animal, platelet deposition spanned a 3-fold range. Platelet deposition was significantly correlated with whole-blood lymphocyte counts and with platelet counts., Conclusions: Individual propensity for arterial thrombosis in pigs is more greatly influenced by blood components than by elements within the arterial wall.

Atherosclerosis manifests as a systemic disease with near global involvement of the named segments of the arterial tree. Acute thrombotic arterial occlusion, however, is not equally distributed. To evaluate intra-individual regional differences in arterial thrombogenicity, we compared (111)In-platelet deposition in porcine carotid and femoral arteries after a standardized crush injury. Within the unidirectional flow conditions of elastic carotid arteries, platelet deposition was more than 3-fold higher compared with predominantly muscular femoral arteries with triphasic arterial flow. To determine the influence of rheology on platelet deposition after crush injury, carotid arteries were transplanted into the femoral position and compared with the paired native carotid and femoral arteries. Similarly, femoral arteries transposed to the carotid position were compared with the paired native carotid artery. In each of these experiments, arterial transposition to a new anatomic location imparts a predilection for platelet deposition indigenous to the new location. In the controlled environment of two high-shear thrombin-independent and -dependent flow chambers, porcine carotid and femoral arterial substrates were indistinguishable from one another with respect to platelet deposition. Regional differences in arterial hemodynamics may account for substantial differences in thrombosis arising from deep arterial injury.

Arterial thrombophilia independent of vascular pathology has not been previously defined either experimentally or epidemiologically. To address the existence of an individual propensity to arterial thrombosis, we exploited a previously developed procedure entailing traumatic (crush) injury of paired porcine carotid arteries for generating platelet-rich thrombi. Porcine carotid arteries were injured bilaterally by serial hemostat crushes. Thrombus generation was monitored by local accumulation of autologous 111In-labeled platelets and Doppler blood flow. Within this cohort of animals of similar age and size, the lowest to the highest responders in thrombus mass spanned a 7-fold range, showing no correlation with shear, platelet or leukocyte count, or plasma concentrations of fibrinogen or von Willebrand factor. However, there was strong intra-individual correlation (r2=0.80; P<0.001) of thrombus deposition between carotid artery pairs. The wide variation in thrombotic response to a standardized stimulus, not accounted for by shear stress or typical hematological variables, appears to be an intrinsic propensity of the individual. The experimental system for thrombus generation is sufficiently quantitative for assessment of variables determining this propensity.

Acute stent thrombosis remains a major concern of coronary stent implantation. Animal studies using stents do not adequately mimic this clinical problem, since stent placement is rarely associated with acute closure. The purpose of this study was to develop and characterize a porcine model of stent thrombosis. Improved understanding through such a model may be useful toward preventing and treating acute stent closure. Whole blood was drawn from domestic crossbred swine one day before study. Platelets were isolated, labeled with 111-In tropolone, and reinjected within 18 hr of the study. Bilateral carotid arteries were exposed, and severe injury induced by a series of mechanical crushes. This method produced histologic injury similar to human coronary angioplasty, with medial disruption and large dissections protruding into the lumen. Stenting was performed in standard fashion with 3.5-mm JJIS stents. Local platelet deposition was measured and recorded as 111-In radioactivity using a miniaturized scintillation detector (Dosimeter Corp.) mounted directly at the artery injury site. This measurement was made in real time at 1-min intervals. Similarly, volumetric blood flow was measured in real time by Doppler flowmeter. Eighteen arteries of nine pigs were studied. In nine arteries from nine pigs, crush injury only was performed and monitored. In the contralateral artery, crush injury was followed immediately by placement of a 3.5-mm Palmaz-Schatz (coronary) stent. Blood flow decreased rapidly following injury in both groups and followed a cyclic pattern. Eight arteries of the crush alone and two arteries of the crush plus stent groups were totally occluded 1 hr after crush. 111-In counts normalized to baseline were significantly higher at 1 hr in both groups compared to baseline; in the stented group, counts were higher than in the unstented group. Blood flow was higher in the stented group than in unstented group for 1 hr. Histopathologic observation of the thrombi forming in both crush-only and crush-stent injuries showed severe medial dissections with obstructing medial flap formation. The thrombi forming in both groups were highly platelet rich. This model of stent and arterial thrombosis showed rapid formation of platelet-rich thrombus, cyclic blood flow variations, and acute occlusion in 20% of cases. Stent placement at arterial injury sites is associated with thrombus that is predominantly platelet rich. Stent placement at injury sites enhances platelet deposition over crush injury alone. Despite greater numbers of platelets, as shown by increased 111-In counts, stenting improved vessel patency. These were likely due to higher volumetric blood flow, continuous deposition, and embolization of labeled platelets.