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Background: Hemophilia A and B are recessive X-linked bleeding disorders characterized by the deficiency in coagulation factor VIII and factor IX. Patients with the severe form of the disease can experience bleeding into the joints and muscle either spontaneously or after provoking events including sporting injuries of different degree. Patients are prone to bleeding in the musculoskeletal system. While most research is focused on hemarthrosis, muscle hematomas are not well studied although being the second most common complication of hemophilia, accounting for 10-25% of diagnosed bleeding episodes. There is limited consensus regarding optimal diagnosis and treatment of muscle hematomas in patients with hemophilia. We are investigating the effect of physical activity on muscle and joint bleeds in mouse models of hemophilia. Aim: The objective of this study was to compare the severity of exercise-induced skeletal bleedings in two mouse models of hemophilia A and B. Methods: Hemophiliac F8-KO and F9-KO mice, aged from 8-12 weeks old, were subjected to either mild or moderate daily treadmill exercise for 5 days. The speed was set at 10 meters per minute for the mild intensity group and 15 meters per minute for the moderate intensity group; and the animals were exercised for 30 minutes daily. Mechanical stimulus with a brush was used to encourage the animals to run. Animals were monitored daily and on day 6 (24 hours after the treadmill exercise) the animals were sacrificed to assess bleeding events. The mice skin was carefully removed and the body was macroscopically evaluated for presence of bleeds. If muscle bleedings were present, they were assigned a score in accordance with a previously published protocol (Tranholm M et al., JTH 2015 Jan;13(1):82-91) with minor modifications. Results: The animals were closely monitored during the study periods, and all completed the study. Normal behaviour and appearances were observed. No external bleeding was recorded. No muscle hematoma was observed in the wild-type C57-strain, thus the group received the bleeding score of 0. For the mild exercise group, the mean muscle bleeding score of F8-KO strain was 5.50 (standard error = 2.81), while the mean muscle bleeding score of F9-KO strain was 1 (standard error: 2). The two-tailed P value between the hemophilia A and B group was significant (p Discussions: The preliminary analysis of our data showed that musculoskeletal bleeding induced by exercise appears to be more severe in hemophilia A mice than hemophilia B. This is in accordance with previous observations that hemophilia B may be clinically milder than hemophilia A. In our study, there appears to be a more significant difference between the hemophilia A and B mice when the mice were subject to milder form of exercise. No statistical significance was found between the two groups when moderate exercise was given. Overall, this exercise model can be a useful tool to study the pathophysiology of musculoskeletal bleeding as well as to evaluate new therapeutic strategies in the prevention and management of muscle hematomas in hemophilia. Figure 1 Figure 1. Disclosures Gross: Leo Pharma: Honoraria; Bayer: Honoraria; BMS-Pfizer: Honoraria; Valeo: Honoraria. Matino: Bayer: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Spark: Other: research grants; Octopharma: Membership on an entity's Board of Directors or advisory committees, Other: research grants and personal fees; Sobi: Membership on an entity's Board of Directors or advisory committees, Other: personal fees.

Cancer is a risk factor for thrombosis, but unlike venous thrombosis, its association with ischemic stroke, and its impact on stroke management and post-stroke outcomes are not well characterized. The primary objective of this study was to measure and compare the risk of ischemic stroke in individuals with a new diagnosis of cancer and those without a history of cancer in two separate matched cohorts based on the absence (Matched Cohort 1) or presence (Matched Cohort 2) of a prior diagnosis of ischemic stroke (Figure 1). Methods: We conducted a population-based matched cohort study of adults ≥18 years using linked clinical and administrative health databases in Ontario, Canada (2010 to 2019). Individuals with a new diagnosis of cancer were matched (1:1) to cancer-free controls by age and sex. Cancer diagnoses were determined using ICD-O-3 diagnostic codes (basal and squamous cell carcinoma and primary central nervous system tumors were excluded). For cancer patients, the index date was the diagnosis date and a corresponding dummy index date was assigned to the matched cancer-free control. The primary outcome was the incidence of ischemic stroke (time to ischemic stroke following index) determined using validated ICD-9 or -10 diagnostic codes from hospitalizations or emergency department visits. Analyses were conducted separately, in parallel, for each cohort. Standardized differences were used to compare the distributions of baseline characteristics. Cumulative incidence function (CIF) curves were generated for ischemic stroke and all-cause mortality. Competing risk methods were used to calculate sub-distribution adjusted hazard ratios (aHR) and 95% confidence intervals (CI) for ischemic stroke, where death was treated as a competing event. Interactions with the timeline at 1.5 years after index were incorporated to assess if the hazard ratio for the main exposure (cancer vs cancer-free) varied before and after the 1.5-year mark. Regression analyses were adjusted using baseline characteristics for which there was imbalance between groups. Results: Matched Cohort 1 included 620,647 individuals with cancer and 620,647 controls. Matched Cohort 2 included 13,924 individuals with cancer and 13,924 controls. Baseline characteristics were generally well balanced (Table 1). Use of antithrombotic medications (prior to index) among individuals ≥66 years was similar between the groups (data not shown). CIF curves for ischemic stroke and mortality are shown in Figure 2. In Matched Cohort 1 (no history of stroke), the risk of ischemic stroke was increased in cancer patients compared to cancer-free controls 1.5 years post-index (aHR 1.40, 95%CI 1.34-1.47). From 1.5 years to 5 years, the risk of ischemic stroke was lower in cancer patients compared to controls (aHR 0.72, 95%CI 0.69-0.74). In Matched Cohort 2 (prior history of ischemic stroke), the risk of ischemic stroke was similar at 1.5 years after the index date in individuals with and without cancer (aHR 1.00, 95%CI 0.88-1.14). From 1.5 to 5 years, the risk of ischemic stroke was reduced (aHR 0.53, 95%CI 0.46-0.62) in cancer patients compared to controls. Conclusions: Compared to cancer-free controls, the risk of ischemic stroke among individuals with a new cancer diagnosis depended on the presence or absence of prior history of ischemic stroke, a novel finding not previously reported. In individuals without a prior history of stroke, those with cancer had a 1.5-fold higher risk of ischemic stroke at 1.5 years compared to controls. In individuals with a prior history of stroke, those with cancer had a similar risk of stroke compared to controls. At 5 years post-index, the risk of ischemic stroke was lower in cancer patients in both cohorts which may reflect high early mortality rates and lower stroke risk among long-term survivors of cancer. The risk of death among cancer patients was highest during the first 1.5 years after the index date in both cohorts. However, the magnitude of the risk increase was higher in individuals without a prior history of stroke (10-fold) compared to those with a prior history of cancer (5-fold). Limitations include unknown causes of death and unavailability of some covariates (e.g. smoking and antithrombotic use in individuals aged Figure 1 Figure 1. Disclosures Siegal: BMS-Pfizer: Honoraria; Leo Pharma: Honoraria; Novartis: Honoraria; Portola: Honoraria; Servier: Honoraria. Carrier: Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Servier: Honoraria; Leo Pharma: Honoraria, Research Funding; Bayer: Honoraria; Sanofi: Honoraria. Gross: Valeo: Honoraria; Leo Pharma: Honoraria; Bayer: Honoraria; BMS-Pfizer: Honoraria.

Factor (F) XII, a key component of the contact system, triggers clotting via the intrinsic pathway, and is implicated in propagating thrombosis. Although nucleic acids are potent activators, it is unclear how the contact system is regulated to prevent uncontrolled clotting. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa and attenuates its capacity to trigger coagulation. To investigate the role of HRG as a regulator of the intrinsic pathway, we compared RNA- and DNA-induced thrombin generation in plasma from HRG-deficient and wild-type mice. Thrombin generation was enhanced in plasma from HRG-deficient mice, and accelerated clotting was restored to normal with HRG reconstitution. Although blood loss after tail tip amputation was similar in HRG-deficient and wild-type mice, carotid artery occlusion after FeCl3 injury was accelerated in HRG-deficient mice, and HRG administration abrogated this effect. To confirm that HRG modulates the contact system, we used DNase, RNase, and antisense oligonucleotides to characterize the FeCl3 model. Whereas DNase or FVII knockdown had no effect, carotid occlusion was abrogated with RNase or FXII knockdown, confirming that FeCl3-induced thrombosis is triggered by RNA in a FXII-dependent fashion. Therefore, in a nucleic acid-driven model, HRG inhibits thrombosis by modulating the intrinsic pathway of coagulation.

The new oral anticoagulants (NOACs), which include dabigatran, rivaroxaban, apixaban, and edoxaban, are poised to replace warfarin for treatment of the majority of patients with venous thromboembolism (VTE). With a rapid onset of action and the capacity to be administered in fixed doses without routine coagulation monitoring, NOACs streamline VTE treatment. In phase 3 trials in patients with acute symptomatic VTE, NOACs have been shown to be noninferior to conventional anticoagulant therapy for prevention of recurrence and are associated with less bleeding. Rivaroxaban and dabigatran are already licensed for VTE treatment in the United States, and apixaban and edoxaban are under regulatory consideration for this indication. As the number of approved drugs increases, clinicians will need to choose the right anticoagulant for the right VTE patient. To help with this decision, this review (1) compares the pharmacologic profiles of the NOACs, (2) outlines the unique design features of the phase 3 trials that evaluated the NOACs for VTE treatment, (3) reviews the results of these trials highlighting similarities and differences in the findings, (4) provides perspective about which VTE patients should receive conventional treatment or are candidates for NOACs, and (5) offers suggestions about how to choose among the NOACs.

Despite the introduction of direct oral anticoagulants (DOACs), the search for more effective and safer antithrombotic strategies continues. Better understanding of the pathogenesis of thrombosis has fostered 2 new approaches to achieving this goal. First, evidence that thrombin may be as important as platelets to thrombosis at sites of arterial injury and that platelets contribute to venous thrombosis has prompted trials comparing anticoagulants with aspirin for secondary prevention in arterial thrombosis and aspirin with anticoagulants for primary and secondary prevention of venous thrombosis. These studies will help identify novel treatment strategies. Second, emerging data that naturally occurring polyphosphates activate the contact system and that this system is critical for thrombus stabilization and growth have identified factor XII (FXII) and FXI as targets for new anticoagulants that may be even safer than the DOACs. Studies are needed to determine whether FXI or FXII is the better target and to compare the efficacy and safety of these new strategies with current standards of care for the prevention or treatment of thrombosis. Focusing on these advances, this article outlines how treatment strategies for thrombosis are evolving and describes the rationale and approaches to targeting FXII and FXI. These emerging anticoagulant strategies should address unmet needs and reduce the systemic underuse of anticoagulation because of the fear of bleeding.

In this issue of Blood , Panicker and colleagues have clarified the role of soluble P-selectin (sP-selectin) in inflammation and cardiovascular disease by showing that monomeric sP-selectin, the form that circulates in plasma, does not promote inflammation or promote coagulation, but the dimeric form of P-selectin does. 1

Introduction: The perioperative management of patients who are taking a direct oral anticoagulant (DOAC) for atrial fibrillation (AF) and require an elective surgery/procedure is uncertain. No studies have addressed the timing of perioperative DOAC interruption and resumption, and if perioperative heparin bridging and coagulation function testing are needed. The Perioperative Anticoagulant Use for Surgery Evaluation (PAUSE) Study hypothesized that a simple, standardized perioperative management strategy, based on DOAC-specific interruption and resumption intervals, that foregoes perioperative heparin bridging and coagulation function testing, is safe for patient care, with associated low rates of major bleeding (1%) and arterial thromboembolism (0.5%). We postulated that this management yields a high proportion of patients (>90%) with a minimal to no DOAC level at surgery/procedure. Methods: PAUSE is a prospective study with 3 parallel DOAC cohorts of patients with AF taking apixaban, dabigatran or rivaroxaban and requiring anticoagulant interruption for an elective surgery/procedure. Patients were managed using a standardized protocol based on DOAC pharmacokinetic properties, procedure-associated bleeding risk (Appendix 1) and creatinine clearance (CrCl). DOACs were interrupted for 1 day before and after surgery for a low bleed risk surgery and 2 days before and after a high bleed surgery; longer interruption was done in patients on dabigatran with a CrCl Results: We enrolled 3007 patients from 23 sites in Canada, the U.S. and Europe (Appendix 4). The patient characteristics were (Figure 2): mean age 72.5 years; 66.1% male; 33.5% high bleeding risk surgery/procedure, with 1257 patients in the apixaban cohort, 668 in the dabigatran cohort and 1082 in the rivaroxaban cohort (Table 1). DOAC interruption and resumption intervals are shown in Table 2. The 30-day postoperative rate (95% CI) of major bleeding was 1.35% (0-2.00) in the apixaban cohort, 0.90% (0-1.73) in the dabigatran cohort and 1.85% (0-2.65) in the rivaroxaban cohort; the rate (95% CI) of arterial thromboembolism was 0.16% (0-0.48) in the apixaban cohort, 0.6% (0-1.33) in the dabigatran cohort and 0.37% (0-0.82) in the rivaroxaban cohort (Table 3). There were 2541 (84.5%) patients with preoperative DOAC levels measured: a level Conclusions: In patients with AF who were taking a DOAC (apixaban, dabigatran, rivaroxaban) and required anticoagulant interruption for an elective surgery/procedure, using a standardized DOAC-specific perioperative management strategy was safe for patient care, with associated low rates of perioperative MB (90% overall; 98.8% at high bleeding risk) had a minimal or no residual DOAC level at the time of the surgery/procedure. PAUSE is the largest study, to date, that addresses how to manage the common problem of perioperative DOAC management. It is likely to have a practice-changing impact and will inform future practice guidelines in perioperative care. Study Funding: CIHR (313156) and the H&S Foundation of Canada (G-14-0006136). Aniara-Hyphen Biomed (assays). Acknowledgments: We thank Drs. Walter Ageno, David Garcia, Lehana Thabane, Wendt Lim, Lori Linkins, William Ristevski, and Demetrios J. Sahlas. Also, Kayla Lucier, Grace Wang, Tara McDougall, and HRLMP and CRLB. Supported by CanVector and REDCap. Disclosures Douketis: Bayer: Other: Advisory Board; Janssen: Consultancy; BMS: Other: Advisory Board; Biotie: Other: Advisory Board; Daiichi-Sankyo: Other: Advisory Board; Boehringer-Ingelheim: Consultancy, Other: Advisory Board, Research Funding; The Medicines Company: Other: Advisory Board; Sanofi: Consultancy, Other: Advisory Board; Astra-Zeneca: Other: Advisory Board; Portola: Other: Advisory Board; Pfizer: Other: Advisory Board. Spyropoulos:Janssen Scientific Affairs, LLC: Consultancy. Carrier:Bayer: Honoraria; Leo Pharma: Research Funding; Pfizer: Honoraria; BMS: Honoraria, Research Funding. Vanassche:Bayer: Consultancy; Boehringer Ingelheim: Consultancy; BMS/Pfizer: Consultancy. Verhamme:Bayer: Honoraria, Research Funding; Medtronic: Honoraria; Portola: Honoraria; Boehringer Ingelheim: Honoraria; Leo Pharma: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Shivakumar:Pfizer: Honoraria; Servier: Honoraria; Bayer: Honoraria; LEO Pharma: Honoraria. Gross:Pfizer: Honoraria; Bayer: Honoraria; LEO Pharma: Honoraria; Servier: Honoraria. Lee:Pfizer: Consultancy, Research Funding; BMS: Research Funding; Servier: Honoraria; LEO Pharma: Consultancy, Research Funding; Bayer: Consultancy, Honoraria. Le Templier:BMS-pfizer: Honoraria. Wu:Leo Pharma: Honoraria; Pfizer: Honoraria; BMS-Pfizer: Honoraria. Coppens:Bayer: Honoraria, Other: Non-financial support, Research Funding; CSL Behring: Honoraria, Other: non-financial support, Research Funding; Uniqure BV: Research Funding. Arnold:Bristol Myers Squibb: Research Funding; UCB: Consultancy; Amgen: Consultancy, Research Funding; UCB: Consultancy; Amgen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Consultancy, Research Funding. Caprini:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Recovery Force: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizor: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Membership on an entity's Board of Directors or advisory committees. Summer:Octapharma: Honoraria. Schulman:Daiichi-Sankyo: Honoraria; Bayer: Honoraria; Sanofi: Honoraria; Boehringer-Ingelheim: Honoraria, Research Funding.

We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWF-independent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre+, Fnflox/flox, Fg/VWF−/−) mice and littermate control (Cre−, Fnflox/flox, Fg/VWF−/−) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti–mouse β3 integrin antibodies but was not affected by anti–mouse GPIbα antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl3-injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1–deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1–deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1–deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1–deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

Abstract 484 Background: Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods: To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (>98%) and platelet pFn (>80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results: We first observed a significantly higher mortality in TKO (25%, P We further found that the mortality rate in Fg/pFn−/− mice was also higher than their Cre- Fg−/− littermates (29%, P We also found that pFn supports hemostasis in VWF−/− mice, although no significant mortality difference was observed (P>0.05). The tail vein bleeding time was longer in VWF/pFn−/− mice than in Cre- VWF−/− littermates (P pFn was also found to support hemostasis in a fibrin-dependent manner. We first demonstrated with TEG that fibrin clot strength was significantly stronger in Cre- littermates than in pFn−/− mice (P Very interestingly, in keeping with our earlier observation in TKO mice, pFn also inhibited platelet aggregation when fibrin was absent. In Fg−/− mice, we found that pFn depletion enhanced gel-filtered platelet aggregation induced by both thrombin and thrombin receptor activating peptide (TRAP, AYPGKF; P Conclusion: Our data demonstrated that pFn is a critical factor for the survival of Fg−/− mice and supports hemostasis in VWF−/− mice via both fibrin-independent and dependent pathways. We clearly showed that fibrin, likely in the form of covalently-linked fibrin-pFn complexes, is required for pFn to support platelet aggregation. Through inhibition of platelet aggregation, non-fibrin-linked soluble pFn may play an important role in the prevention of excessive thrombus formation at the site of vessel injury and thus maintains blood circulation. pFn is therefore likely a crucial supportive factor in hemostasis (for afibrinogenemic and VWD patients), and an important regulator in thrombosis. Disclosures: No relevant conflicts of interest to declare.

Introduction: Microparticles derived from leukocytes contribute to fibrin formation at thrombi in vivo and factor VIII-deficient (FVIII) mice treated with an agent that elevates their microparticles have decreased bleeding. A novel therapy for hemophilia patients with inhibitors is needed. We evaluated whether microparticles generated in vitro could improve hemostasis in FVIII mice. Methods: Mouse CD11b positive monocytes, isolated by MACS, or cultured monocytic cells (WEHI274.1) were treated with the calcium ionophore A23187. The resulting microparticles (isolated by differential centrifugation, and defined as CD18 positive events less than 1 μm diameter) or PIPES buffer were infused intravenously into FVIII-deficient mice (B6.129S4-F8tm1Kaz) or control wild type B6.129 mice prior to evaluation. The amount of platelets in laser-generated thrombi in cremaster muscle arterioles was evaluated using high-speed intravital fluorescence microscopy. The amount of hemoglobin shed from a 2 mm tail tip amputation measured blood loss. Results: Infusion of MPs at doses above 1000/g resulted in the death of wild type mice; FVIII-deficient mice tolerated MPs at doses up to 4000/g. Blood loss after tail clip in FVIII-deficient mice was 6-fold higher than blood loss from wild type mice. Blood loss after tail clip in FVIII-deficient mice was reduced to normal after the infusion of MPs at concentrations as low as 400/g. MPs, at 400/g, from CD11b positive cells isolated from wild type, FVIII-deficient mice or PSGL-1-deficient mice all similarly reduced blood loss after tail clip in FVIII-deficient mice. The biological half life of MP effect on tail-bleeding was 3 hours. Platelet accumulation in cremaster arteriolar thrombi was impaired in FVIII-deficient mice. Infusion of MPs at a concentration of 1000/g normalized platelet accumulation, but infusion of MPs at a lower concentration (400/g) did not. Conclusion: Abnormal hemostasis in FVIII-deficient mice can be temporarily reversed by the infusion of in vitro generated monocyte-derived MPs, including MPs derived from monocytes from FVIII-deficient or PSGL-1-deficient mice. The dose whereby MPs normalize FVIII-deficient mice is different between the hemostasis and thrombosis models. To explore whether P-selectin at injuries is required for the effect of MPs, we have generated by cross-breeding FVIII/P-selectin double deficient mice. These mice are born at expected mendelian frequency. Two of 20 male FVIII/P-selectin double deficient mice had spontaneous bleeding at 8 weeks of age, one in the thigh and one from the ear. FVIII/P-selectin double deficient mice also have prolonged tail bleeding times, which will serve as a model for testing the P-selectin targeting of microparticles.

Background: Histone H1.3 suppresses tumor growth of leukemic cells in vitro, ex vivo and in animal models. To evaluate toxicity and efficacy of ONCOHIST, recombinant human histone H1.3 (rhH1.3) in patients suffering from acute myelogenous leukemia, we initiated a phase-I/II dose escalation study. Methods: This was an open-label unicentric phase I/II study. Nine patients suffering from relapsed or refractory acute myeloid leukemia (median age: 69 years, range 49 to 83 years) with a life expectancy of at least one month who were unable or unwilling to receive curative chemotherapy without major organ dysfunction were eligible for the trial. Exclusion criteria were presence of HIV, HBV, or HBC infection, heparin treatment during the 2 weeks before enrolment, and medical conditions known to potentially interfere with rhH1.3 treatment such as rheumatoid arthritis or SLE, as well as circulating anti-histone H1.3 antibodies. One treatment course consisted of 9 applications in 3 weeks. 3 patients were treated at each dose level. Starting dose level was 245 mg/m2 and in the absence of dose-limiting toxicities, the dose was increased to the next higher dose level (392 and 628 mg/m2 rhH1.3) in the third week of the treatment course. Primary endpoints of the study was the definition of the maximal tolerated dose and dose-limiting toxicities of the study drug. Results: To date, 9 patients have been treated and all patients are evaluable for toxicity and efficacy. No side-effects were observed except for one atrial fibrillation under infusion of rhH1.3, which was considered not to be related to the study drug. All patients completed one course of therapy (9 applications), and one responding patient received a second course without side effects. No dose-limiting toxicities were observed and the maximal tolerated dose has not been reached at 628 mg/m2. With respect to efficacy, the formal criteria of response were not met in any of the 9 patients. However, two patients had a temporary increase of their platelet counts while on therapy, and in one patient platelet counts raised from 22 000/mm3 to 100 000/mm3, remaining stable for 3 months. Two patients achieved a reduction of blasts upon treatment. Conclusion: rhH1.3 is well tolerated at the doses treated so far. While the achieved serum levels are still below the growth-inhibiting concentrations in vitro, first clinical effects have been observed. Additional patients and higher dose levels are needed to delineate the efficacy and toxicity profile of rhH1.3 for the treatment of acute myelogenous leukemias.

Background: In venous thrombi, urokinase associated with leukocytes activates plasminogen to initiate endogenous fibrinolysis. Leukocytes also interact with arteriolar thrombi. Objectives: We hypothesized that urokinase plasminogen activator (uPA) activity would also be delivered to arteriolar thrombi by leukocytes. Methods: Using established techniques of high-speed intravital fluorescence microscopy to observe the cremaster muscle of mice, we measured platelet and fibrin accumulation in thrombi generated by a laser injury to arterioles. The accumulation of platelets in a thrombus is quantitated by measuring the fluorescence from anti-CD41 antibody tagged with a fluorescent secondary antibody and the presence of fibrin is detected using a fluorescence-tagged anti-fibrin antibody (T2G1). Results: In C57BL/6 wild type mice, fibrin increased to a maximum at about three minutes after thrombus formation and then decreased, such that by eight minutes after thrombus formation fibrin is present at 34% of its maximal value. When wild type mice are pretreated with epsilon-aminocaproic acid (16 mg/kg IV), the decrease in thrombus fibrin content was less (to 54% of maximal 8 minutes after thrombus formation), implying that the fibrin loss is at least partially the result of plasmin activity. Thrombus fibrin accumulation in tissue-plasminogen activator deficient mice (Plat) was greater, peaking at 229% the amount found in wild type mice, but the amount of fibrin present at eight minutes after thrombus formation was not dissimilar from wild type mice (45% ± 5% v. 34% ± 3%, P=0.09). In uPA-deficient mice (Plau) and uPA-receptor-1-deficient mice (Plaur), the amount of maximal thrombus fibrin was 189% and 273% that of fibrin content in wild type mice. Also, although thrombus fibrin did decrease over time in Plau and Plaur mice, it decreased to only 51% and 48% of maximal (each were P < 0.005 v. wild type) at eight minutes after thrombus formation. When wild type leukocytes, isolated from blood by differential centrifugation and sucrose density gradient, were transfused into Plaur mice, thrombus fibrin loss was restored to wild type levels (38% of maximal at 8 minutes after thrombus formation, P=0.36 vs. wild type, P=0.016 vs. Plaur), implying that transfused wild type leukocytes, which express uPA-receptor-1, can rescue fibrinolysis in uPA-receptor-1-deficient mice. Conclusions: These results confirm that endogenous fibrinolytic activity in vivo occurs soon after arteriolar thrombus formation. The plasminogen activators that are required for this fibrinolysis to occur are uPA, and the uPA-receptor-1 on leukocytes. These findings are compatible with the hypothesis that leukocytes can deliver plasminogen activator activity to arteriolar thrombi to initiate endogenous fibrinolysis.

Background: Statins are well recognized to effect a lowering of plasma lipoproteins and this may be beneficial in cardiovascular disease. Objectives: We investigated whether statins may reduce the burden of atherothrombotic cardiovascular disease independently of their known effects on plasma lipoproteins. Methods: Wild type C57BL/6 mice were fed atorvastatin, at a dose of 33 mg of atorvastatin per kg of standard chow. Controls received the same chow, but without the drug. After 14 days, we induced thrombi within arterioles of the cremaster muscle by laser injury and, using intravital microscopy, quantified platelet accumulation within each thrombus by measuring the fluorescence from anti-CD41antibody with a fluorescent-tagged secondary antibody. Results: Mean platelet accumulation in thrombi was slightly, but significantly, lower at early time points in atrovastatin-fed mice compared with thrombi generated in untreated control mice i.e. one minute after arteriolar injury, area under the curve analysis revealed atorvastatin-fed mice to have 15% less platelet accumulation within thrombi compared to controls (Mann-Whitney test: P = 0.02); thereafter, no difference was observed. To determine whether this effect may be directly related to altered platelet function, we devised a novel method to study platelet accumulation within laser-induced arteriolar thrombi: Platelets were isolated from two mice using differential centrifugation and gel chromatography. The platelets from one mouse were labeled with DiI, while those from the other mouse were labeled with DiO. An equal proportion of labeled platelets were simultaneously transfused into a third mouse. The arrival and departure of individual green-fluorescent (DiI) and red-fluorescent (DiO) platelets within each thrombus were analyzed. This dual-population dual-label technique revealed that platelets from atrovastatin-treated mice accumulated in thrombi half as much as control platelets; this occurred in thrombi generated in both atorvastatin-treated and control mice. Because analysis is independent of the degree of injury, which is variable in the laser-induced model, this technique represents a sensitive new method to compare platelet accumulation among different platelet populations. Some of atorvastatin’s beneficial effects may involve the nitric oxide pathway. S-nitrosylation of N-ethylmaleimide-Sensitive Factor (NSF) inhibits platelet granule secretion, which may reduce platelet accumulation in a thrombus. Accordingly, we compared the amount of S-nitrosylation of NSF in platelets from atorvastatin-treated versus untreated control mice. Platelet proteins were immunoprecipitated with a rat polyclonal anti-conjugated NO-L-cysteine antibody. Western blot testing with anti-NSF revealed more product (normalized for the amount of platelet protein)in platelets from atorvastatin-treated mice than in control platelets. Hence, treatment of mice with atorvastatin may yield platelets that express higher amounts of S-nitrosylated NSF, impairing their ability to accumulate within thrombi. Conclusions: These findings enhance our understanding of other possible mechanisms by which statins effect a rapid reduction in the progression of atherothrombosis.

Background: A phase III trial of antithrombin (AT) in sepsis showed benefit only in patients who did not receive concurrent heparin. We have shown that some of the beneficial effects of AT on leukocyte-endothelial (LE) interactions in sepsis models requires the activity of heparan sulfate 3-O-sulfotransferase-1 (3-OST-1), the enzyme that is responsible for the rate-limiting step in the biosynthesis of anticoagulant heparan sulfate. Fondiparinux (FOND) is a pharmacological agent that mimics the anticoagulant heparan sulfate pentasaccharide sequence synthesized by 3-OST-1. Objectives: We hypothesized that FOND treatment would alter LE interactions in sepsis models in mice, and AT effects in these models. Methods: Using intravital microscopy, we evaluated the effects of proinflammatory stimuli on leukocyte rolling and firm adhesion in post-capillary venules, and extravasation, into the cremaster muscle in live C57BL/6 mice. Human AT (0.25 U/g Thrombate III), and/or heparanoid or vehicle was infused intravenously prior to proinflammatory stimuli. Venules were chosen such that in all comparisons, the average diameter (range 20 to 50 μm) and shear rate (range 200 to 800 s−1) was not different between treatment groups. The number of leukocytes rolling past a defined vessel point was expressed as leukocyte rolling flux, which normalizes for leukocyte count and flow rate. The number of firmly adherent leukocytes (stationary for 30 s) was normalized for the area of vessel wall analyzed. The number of extravasated leukocytes was normalized for the area of tissue analyzed. Results: AT treatment decreased leukocyte rolling flux (23 vs. 15%, P=0.01) induced by an injection of lipopolysaccharide (LPS at 1 μg i.p.) 2 h before observation. However, in this model, AT did not inhibit the number of firmly adherent leukocytes. When mice were pretreated with FOND (0.1 μg/g), the leukocyte rolling flux was increased compared to without heparanoid (22 vs. 25%, P Conclusions: These studies further our knowledge of how the anticoagulant pentasaccharide sequence, mimicked by fondiparinux and lacking in 3-OST-1 deficient mice, is important in leukocyte recruitment that occurs in response to inflammation. This will allow for more rational use of mediators of this pathway such as AT in sepsis.

Antithrombin (AT) exhibits anti-inflammatory properties that reduce mortality in sepsis models. We examined the effects of AT on leukocyte-endothelial interactions in sepsis by using intravital microscopy to measure leukocyte rolling and firm adhesion in post-capillary venules of the cremaster muscle in live mice. Human AT (0.25U g−1 i.v. of Thrombate III, Bayer Corporation) or vehicle was infused prior to proinflammatory stimuli. We evaluated venules with diameters between 20 and 50 μm and shear rates between 200 and 800 s−1. The average diameter and shear rate was not different between treatment groups. The number of leukocytes rolling past a defined vessel point was expressed as leukocyte rolling flux, which normalizes for leukocyte count and flow rate. The number of firmly adherent leukocytes (stationary for 30 s) was normalized for the area of vessel wall analyzed. We evaluated C57BL/6 mice in three inflammatory models. When inflammation was caused by surgical isolation of the cremaster, AT decreased leukocyte rolling flux (28±2 vs. 21±2%, P=0.02) but not the number of firmly adherent leukocytes (41±5 vs. 51±7 per μm2, NS). In contrast, AT did not effect leukocyte rolling flux when inflammation was induced by tumor necrosis factor alpha (0.5 μg intrascrotally). Thus in this model, either pro- and anti-inflammatory effects are balanced or leukocyte rolling involves other interactions that are AT insensitive. Similarly, AT did not affect leukocyte rolling flux induced by an injection of lipopolysaccharide (LPS at 1 μg i.p.) 8 h before observation. However, in this model, AT did inhibit the number of firmly adherent leukocytes (72±6 vs. 39±6 per μm2, P We then tested if these anti-inflammatory effects require a heparan sulfate proteoglycan (HSPG) based AT receptor. We used Hs3st1−/− mice, which lack the isoform of heparan sulfate 3-O-sulfotransferase that is predominantly responsible for the biosynthesis of anticoagulant HSPGs, which bind AT. Although healthy without challenge, 2 of 2 Hs3st1−/− mice died 6 h after LPS administration (1 μg i.p.), whereas none of 18 wild-type (WT) mice (P Thus AT can decrease specific leukocyte-endothelial interactions that occur in sepsis, but the effects are highly model dependent, possibly involving a balance of AT’s pro- and anti-inflammatory activities. HSPGs, on cells other than leukocytes, mediate AT anti-inflammatory activity, as LPS- treated mice lacking this AT receptor have enhanced lethality and exhibit enhanced leukocyte firm adhesion that is augmented by AT treatment.

Statins, inhibitors of 3-Hydroxy-3-methylglutaryl coenzyme A reductase, are known to effectively reduce plasma cholesterol and reduce the mortality and morbidity of atherosclerotic disease. Recent investigation suggests that statins influence vascular biology through mechanisms other than decreased plasma cholesterol. Statins can inhibit the activation of the beta-2 integrin CD18 by inhibiting the signaling molecule RhoA and can block LFA-1 (an integrin that pairs CD11a with CD18 on lymphocytes) from binding its ligand ICAM-1. We have previously described leukocyte (largely granulocytes) rolling on arterial thrombi and leukocyte firm adhesion to the thrombus coincident with CD18 activation. We therefore hypothesized that statins can block leukocyte firm adhesion to a thrombus by inhibiting the activation of CD18 in vivo. A placebo controlled experiment measured the leukocyte thrombus interaction in C57/Bl6 mice treated with atorvastatin (Lipitor) for 14 days prior to in vivo experimentation. The six mice in the atrovastatin group received a diet prepared by Dyets.com containing 0.2 mg of atorvastatin in each gram of food. The six mice in the control group received a normal mouse diet without the drug. All mice consumed approximately 5 g of their respective food each day. We monitored the weight of the mice and of the food to ensure that the mice consumed their diet and that they maintained a consistent weight. The mice were anesthetized via an intraperitoneal injection and the jugular vein was cannulated to maintain anesthesia throughout the procedure. Exteriorization of the cremaster muscle that surrounds the testicle allowed visualization of arterioles. A thrombus was induced with a MicroPoint LASER attached to a microscope. There appeared to be no difference in thrombus formation between the two groups. We observed rolling and firmly adhering leukocytes on the thrombus for 45 minutes after injury. In each mouse, on average three thrombi were induced in arterioles that ranged from 26 to 76 μm in diameter (18 thrombi in control group, 16 thrombi in atorvastatin group). At the end of the experiment blood was obtained from each mouse by cardiac puncture for white blood cell count by hemacytometer. There was no significant difference (P=0.37) in the white blood count for the mice in the different groups of the experiment (see Table). There was no significant difference (P=0.22) between the groups in the number of rolling leukocytes observed suggesting similar thrombus size. Leukocyte firm adhesion to the thrombus was defined as a cell that stuck to the thrombus for more than two minutes. Significantly fewer leukocytes (P=0.019) firmly adhered to a thrombus in the atorvastatin group than in the control group. Thus, atorvastatin reduced the number of firmly adherent leukocytes to a thrombus. Because granulocytes lack CD11a, we propose that atorvastatin inhibited the activation of CD18 by inhibiting the signaling molecule RhoA. The observation that statins can influence the dynamic leukocyte thrombus relationship broadens our understanding of the mechanisms by which statins prevent and treat atherosclerosis. Control Atorvastatin Mean±SEM WBC Count 3,948,303±296,590 4,687,121±371,291 Total Rolling Leukocytes 493±104 341±56 Total Firmly Adherent Leukocytes 4.8±3.3 2.3±2.5