Tissue factor, tissue pathway factor inhibitor and risk factors of atherosclerosis in patients with chronic limbs ischemia: preliminary study.

AIM: Thrombus formation plays a critical role in pathogenesis of cardiovascular complications in atherosclerotic peripheral arterial occlusive disease (PAOD). Tissue factor (TF) initiates the clotting cascade and is considered an important regulator of hemostasis and thrombosis. TF activity is regulated by TF pathway inhibitor (TFPI). The aim of our study was to evaluate plasma levels of the TF, TFPI and their relation to coagulation system and various other risk factors of atherosclerosis in patients with chronic limbs ischemia. METHODS: Plasma TF, total TFPI, truncated TFPI, full-length TFPI were assessed by ELISA using commercially available kits (IMUBIND Tissue Factor; Total TFPI; Truncated TFPI ELISA Kit; American Diagnostica Inc. Stamford) in 62 claudicant patients with PAOD and 20 healthy controls. RESULTS: We observed statistically higher levels of TF (94+/-52 pg/mL), total TFPI (43+/-8 ng/mL), and truncated TFPI (22+/-7 ng/mL) in patients with PAOD compared to healthy individuals (TF: 66+/-15 pg/mL; total TFPI: 36+/-4 ng/mL; truncated TFPI: 14+/-5 ng/mL). Full-length TFPI (20+/-4 ng/mL) is lower in patients with PAOD than in controls (23+/-5 ng/mL). The study indicated a positive correlation between TF and truncated TFPI (r=0.34), total TFPI and full TFPI (r=0.5), total TFPI and truncated TFPI (r=0.83) in patients with PAOD, and negative correlation between full TFPI and truncated TFPI (r=-0.65) in the control. CONCLUSION: Elevated levels of TF, disorders of balance between full-length TFPI and truncated TFPI as well as significantly increased truncated TFPI level in patients with PAOD can be independent risk factors of atherosclerotic complications.

Platelet characteristics associated with coronary artery disease.

BACKGROUND/OBJECTIVE: To test the hypothesis that circulating platelets display evidence of interactions with atherogenesis, platelet capacity to express P-selectin and propensity for spontaneous microaggregation in vitro were measured in samples from normal donors (N), patients with asymptomatic advanced coronary calcification (CC) or acute coronary syndromes (AC). To measure the effect of angioplasty on platelet function, samples obtained before, 30 min after and 24 h after angioplasty were compared. PATIENTS/METHODS: Platelet P-selectin was measured after maximal stimulation with thrombin. Microaggregation was measured as a platelet count deficit in citrate-anticoagulated platelet-rich plasma (PRP) relative to EDTA-anticoagulated blood. RESULTS: P-selectin expression was significantly lower for platelets from patients with either AC or CC compared to normals. In addition, platelets from AC and CC patients have a significantly greater propensity to form microaggregates in citrate anticoagulant. After angioplasty, the PRP-platelet count decreased transiently. CONCLUSION: Both acute unstable and chronic stable coronary disease are associated with an increased share of platelets unable to express P-selectin and an increased share of platelets that microaggregate in citrate anticoagulant. The genesis of these platelet characteristics is not fully explained by focal acute arterial injury and may reflect exposure to systemic atherosclerosis or the atherogenic process.

Inhibition and reversal of platelet-rich arterial thrombus in vivo: direct vs. indirect factor Xa inhibition.

BACKGROUND/OBJECTIVE: The efficacy of a direct factor (F)Xa inhibitor, ZK-807834, was compared with indirect inhibition by enoxaparin for inhibition and deaggregation of acute platelet-rich thrombi in a well-characterized porcine carotid injury model. METHODS: A crush injury was performed on a randomly chosen carotid artery and the thrombus allowed to propagate for 30 min. Pigs then received intravenous drug for 35 min: ZK-807834-Dose 1 (40 microg kg(-1) bolus + 1.5 microg kg(-1) min(-1) infusion, n=6); ZK-807834-Dose 2 (20 microg kg(-1) bolus + 0.75 microg kg(-1) min(-1) infusion; n=6); enoxaparin (1 mg kg(-1) bolus; n=6); or saline (n=6). Five minutes after drug initiation, the contralateral artery was injured. Thrombus size was monitored by scintillation detection of autologous 111In-platelets. RESULTS: The prothrombin time ratio was 2.2 +/- 0.1; 1.4 +/- 0.3; 1.2 +/- 0.9 and 1.1 +/- 0.2, respectively. ZK-807834-Dose 1 significantly inhibited carotid platelet deposition (525 +/- 226 x 10(6) cm(-2); P = 0.008), whereas ZK-807834-Dose 2 (2325 +/- 768) and enoxaparin (1236 +/- 383) were not different from saline (2776 +/- 642). Thrombus deaggregation was greatest for animals receiving ZK-807834-Dose 1 (473 +/- 185). Neither ZK-807834-Dose 2 (1588 +/- 480) nor enoxaparin (1618 +/- 686) was different from saline control (2222 +/- 598). CONCLUSIONS: Direct FXa inhibition with ZK-807834, at a prothrombin time ratio of 2.2, effectively inhibits thrombosis and promptly deaggregates thrombi induced by arterial injury. In contrast, indirect FXa inhibition with enoxaparin was ineffective.

Fibrinogen, fibrin and crosslinking in aging arterial thrombi.

The assumption that fibrin and crosslinked fibrin impart irreversibility to arterial thrombi is explored with procedure developed for measuring changes in platelet function, morphology and fibrinogen metabolism in aging occlusive thrombi, in which the condition of stasis is imposed uniformly. Arterial thrombi containing autologous (111)In labeled platelets were generated in vivo by bilateral mechanical injury of porcine carotid arteries. Vessels containing the platelet-rich thrombi were harvested and incubated intact (37 degrees C) for intervals ranging from 30 min to 12 h. The isolated vessels were then bisected and agitated in culture medium containing tick anticoagulant and hirudin for 60 min. Disaggregated platelets were evaluated for yield (from (111)In radioactivity) viability (dense body ATP secretion) and morphology (electron microscopy). Western analysis of fibrin(ogen) in thrombus extracts was performed using anti-fibrinogen Bbeta- and gamma-chain monoclonal antibodies for thrombi at each time point. A stable recovery of nearly 50% of platelets was observed during 12 h of thrombus aging. As thrombi aged, viability of disaggregated platelets gradually decreased with platelet necrosis the predominant feature beyond 6 h. By western analysis of thrombus extracts, nearly 50% of fibrinogen was cleaved to fibrin and extensively crosslinked within 30 min of injury with no evidence of fibrinolysis. With the exception of a declining proportion of gamma-monomer, these features remain relatively constant during 12 h of thrombus maturation. It is concluded that neither fibrin nor crosslinked fibrin are dominant factors imparting cohesion within platelet thrombi. Furthermore, under conditions of complete arterial occlusion imposed by this experimental design, there is no evidence of endogenous fibrinolysis.

Thrombophilia differences in cerebral venous sinus and lower extremity deep venous thrombosis.

OBJECTIVE: To characterize differences in the prevalence of thrombophilic variables in a large cohort of patients with cerebral venous sinus thrombosis (CVST) and lower extremity deep vein thrombosis (DVT). METHODS: An inception cohort of individuals was identified with first lifetime incident CVST between 1995 and 2005 for whom comprehensive thrombophilia testing was available. To test the hypothesis that thrombophilia prevalence differs with respect to thrombus location, test results were compared to a randomly selected group of patients with lower extremity DVT with comprehensive thrombophilia testing. RESULTS: During this time period, 163 patients with CVST were identified who underwent comprehensive thrombophilia testing. Thrombophilia results were abnormal in 29% including anticardiolipin antibodies (17%), heterozygous factor V Leiden (10%), and heterozygous prothrombin G20210A mutation (n = 14/122; 11%). The prothrombin mutation was more than twice as common in patients with CVST (p = 0.04). Activated protein C resistance, factor V Leiden, and protein C deficiency were more common in patients with DVT (p < 0.05 for each comparison). The anticardiolipin antibodies in patients with CVST were primarily low titer IgM isotype. CONCLUSION: The prevalence of selected thrombophilia factors differs comparing patients with cerebral venous sinus thrombosis and deep vein thrombosis. These differences may offer insights into mechanisms governing the geographic distribution of venous thrombosis.

Cerebral venous sinus thrombosis: Incidence of venous thrombosis recurrence and survival.

OBJECTIVE: To determine whether treatments guidelines for lower extremity venous thrombosis (DVT) could be applied to patients with cerebral venous sinus thrombosis (CVST), the rates of recurrent venous thrombosis and survival for these two diseases were compared. METHODS: The authors studied all patients diagnosed with CVST at the Mayo Clinic between 1978 and 2001. Survival and recurrent venous thrombosis rates (cerebral or noncerebral) were compared with those from patients with DVT. Survival rates were also compared with white US residents. RESULTS: One hundred fifty-four patients (age 40 +/- 19 years) were included (58% women). Warfarin, prescribed in 50% of patients, was continued for an average of 9 months. During a mean of follow up of 36 +/- 47 months (464 patient-years), 20 patients experienced 23 recurrent venous thrombi for an event rate of 5.0/100 patient-years. This recurrence rate was similar to patients with lower extremity DVT (3.8/100 patient-years). Mortality rates were lower for CVST (2.8/100 patient-years) compared with DVT (6.2/100 patient-years; p = 0.001) patients but higher than expected for white US residents (p = 0.001). Increasing age and active malignancy were the only predictors of poor survival. Neither recurrent thrombosis nor survival was influenced by warfarin therapy. CONCLUSIONS: The likelihood of recurrent venous thrombosis is similar after cerebral venous sinus thrombosis (CVST) and lower extremity deep venous thrombosis (DVT). Compared with DVT, survival rates are higher following CVST but are adversely influenced by malignancy and older age.

Videoendoscopic thoracic aorta-to-femoral artery bypass in the pig.

BACKGROUND: The thoracoscopic approach to the aorta has the advantages of easy aortic dissection, excellent inflow, improved exposure in the thorax without insufflation, and ability to employ both laparoscopic and traditional instruments. Our aim was to develop a thoracoscopic technique for descending thoracic aorta-to-femoral artery bypass (TAFB) in the pig that results in acceptable short-term survival and graft patency. MATERIALS AND METHODS: Thoracoscopic TAFB was performed in 11 pigs. Using two-lung ventilation, the animals were placed in a 45 degrees left lateral semidecubitus position. A fan lung retractor, two dissecting ports, intercostal artery loops, and camera were placed through five 10- to 20-mm thoracoscopic incisions. After aortic dissection, an 8-mm graft was passed through a retroperitoneal tunnel. Rumel tourniquets were used for aortic occlusion after placement of a shunt. End-to-side endoscopic anastomosis was completed with knots tied extracorporeally. The left femoral anastomosis was completed under direct vision. Duplex ultrasound of the graft was done on postoperative days 1, 3, and 7. RESULTS: Thoracoscopic TAFB was completed in all animals. Mean aortic anastomosis time was 57 min (range, 34-145); and mean cross-clamp time, 74 min (range, 53-155). Mean operative time was 310 min; the first six operations lasted longer than the last five (338 min vs 276 min, P < 0.04). Average blood loss was 611 ml (range, 250-1300). Two animals died due to anesthetic complications. One (11%) of the nine surviving pigs died on day 2 due to bleeding. Complications were paraplegia in one (11%) and graft thrombosis in another (11%). CONCLUSIONS: Videoendoscopic TAFB can be completed in pigs with acceptable short-term patency and survival. Further experience in thoracoscopic techniques can make TAFB a feasible and low-risk option for selected patients with aortoiliac occlusive disease.

Platelet storage solution improves the in vitro function of preserved platelet concentrate.

BACKGROUND AND OBJECTIVES: Stored platelets develop biochemical lesions, manifest as depressed haemostatic function, clot retraction and wound healing. ViaCyte trade mark, a proprietary experimental preservative solution (comprising D-ribose, D-glucose, Hanks solution, Hepes solution, bovine serum albumin, tic anticoagulant peptide and sterile water), was tested in comparison with the presently accepted storage solution, citrate-dextrose-phosphate-plasma (CDP-P), to evaluate its ability to preserve platelet function during storage. MATERIALS AND METHODS: Platelets stored in ViaCyte and platelets suspended in CDP-P were transferred to polypropylene tubes with PL732 covers and analysed for adenine nucleotide levels (ATP molecules), in vitro agonist-mediated P-selectin expression and aggregation. RESULTS: After 5 days of storage at room temperature, 12.2% of platelets stored in ViaCyte exhibited P-selectin expression at rest, and 64.2% exhibited P-selectin expression upon activation with thrombin challenge, an increase of 52%. Platelets stored in CDP-P exhibited 44.4% P-selectin expression at rest, suggesting significant activation during storage, and thrombin stimulation resulted in P-selectin expression of 47.9%, an increase of only 2.5% (P< or =0.002, untreated vs. treated). ViaCyte also maintained ATP levels throughout the storage period, while these levels became depressed in platelets stored in CDP-P (P< or =0.02, untreated vs. treated). Storing platelets in the experimental preservative solution maintained their ability to aggregate, while control platelets lost their ability to aggregate in response to agonist. CONCLUSIONS: ViaCyte appears to protect platelets during storage, reflected by a low level of induced lesions. Platelets stored in ViaCyte maintain energy levels at their resting state, which preserves their ability to aggregate and secrete granule contents, and ensures the availability of additional platelets for activation upon in vitro challenge.