Krzysztof Karnicki

Factors Contributing to Individual Propensity for Arterial Thrombosis

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.

Immunohistochemistry of thrombi following iliac venous stenting: A novel model of venous thrombosis

Stenting has become a common intervention for venous occlusive disease. Little is known regarding the composition of venous thrombi complicating stent placement. The optimal design of antithrombotic agents in this setting requires this knowledge. Quantitative immunohistochemistry was undertaken to define the platelet, fibrin(ogen) and leukocyte composition and spatial orientation of venous thrombi following percutaneous iliac stent placement in pigs. Venous stent thrombus size was measured by weight and scintillation detection of autologous (111) In-platelets. Thrombi were divided in segments (cephalad to caudad), sectioned and stained with monoclonal anti-platelet glycoprotein Ib or polyclonal anti-fibrin(ogen) fluorescent antibodies. Thrombus platelet content was 100-fold greater than paired whole blood samples. The caudal-most segments contained platelet-rich aggregates (p < 0.05) with abundant leukocytes (p < 0.0001) relative to more cephalad segments. Platelet and fibrin(ogen) content varied over an eight-fold range between segments but were directly correlated with each other (r = 0.77; p < 0.0001). The platelet co-localization with fibrin(ogen) is consistent with the phospholipid dependence of prothrombin activation. The abundance and caudal distribution of platelet-leukocyte aggregates indicate their preferential accretion from flowing blood early in the genesis of venous stent thrombi. These may represent novel cellular targets for the prevention and treatment of venous thrombosis.

Iliac Venous Stenting. Antithrombotic Efficacy of PD0348292, an Oral Direct Factor Xa Inhibitor, Compared With Antiplatelet Agents in Pigs

Objective—The clinical use of venous stents is increasing dramatically. Although antiplatelet agents are required for arterial stent patency, optimal thrombo-prophylaxis after venous stenting remains undefined. To address this issue, PD0348292, a direct Factor Xa inhibitor, was compared with antiplatelet therapy in a porcine venous stent model. Methods and Results—Four hours before stent deployment, pigs (n=5 to 6 per group) received oral PD0348292 at 0.4, 0.9, 4.3 mg/kg, or 0.4 mg/kg plus aspirin (325 mg). Aspirin, clopidogrel (75 mg), aspirin plus clopidogrel, or vehicle (n=10) were administered daily for 2 days before the procedure. Two hours after stent placement, thrombi were quantified by autologous 111In-platelet content and weights. Thrombus weight and platelet deposition were significantly reduced by PD0348292 at 0.4 (49±79 mg and 110±145×106/cm2), 0.9 (5±6 mg and 107±128×106/cm2), 4.3 mg/kg (0±0 mg and 87±125×106/cm2), and PD348292 plus aspirin (20±40 mg and 157±70×106/cm2) compared with vehicle (402±226 mg; 584±454×106/cm2). Despite prolonging bleeding times and inhibiting platelet aggregation, neither aspirin (567±683 mg and 533±622×106/cm2), clopidogrel (404±349 mg and 178±101×106/cm2), nor aspirin plus clopidogrel (247±261 mg and 231±266×106/cm2) significantly decreased stent thrombosis. Conclusions—PD0348292 completely inhibited thrombosis after venous stenting. Platelet accretion in these venous thrombi appear to involve pathways distinct from arachidonate metabolism or ADP P2Y12 receptor activation.

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

Summary.  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 µg kg−1 bolus + 1.5 µg kg−1 min−1 infusion, n = 6); ZK‐807834‐Dose 2 (20 µg kg−1 bolus + 0.75 µg 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 × 106 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.

Comparison of PD0348292, a selective factor Xa inhibitor, to antiplatelet agents for the inhibition of arterial thrombosis

The objective of this study was to determine if orally-administered PD0348292, a direct specific factor Xa inhibitor, inhibits thrombosis following porcine carotid arterial injury comparably to aspirin or clopidogrel alone or in combination. We further sought to determine whether the antithrombotic efficacy in vivo could be predicted using an ex-vivo perfusion chamber. Oral treatments included: PD0348292 (0.4, 0.9, or 4.3 mg/kg); PD0348292 (0.4 mg/kg) plus aspirin (325 mg); aspirin; clopidogrel (75 mg); aspirin plus clopidogrel; or vehicle (n = 6-10/group). Aspirin and clopidogrel were administered 27 and four hours pre-injury and PD0348292 or vehicle was administered four hours pre-injury. Both carotid arteries were crush-injured, and thrombus was measured by detection of (111)In-platelets over 30 minutes. Prior to injury, the antithrombotic efficacy was assessed by ex-vivo perfusion chamber platelet deposition. PD0348292 produced dose-dependent prothrombin time (0.9- to 2.9-fold) and aPTT (1.4- to 2.5-fold) prolongations. Bleeding times were significantly prolonged in each active drug group compared to vehicle, but were not significantly different between drug groups. PD0348292 significantly inhibited arterial platelet deposition (x10(6)/cm(2)) at 4.3(549 +/- 1,066), 0.9 (399 +/- 162) and 0.4 mg/kg (531 +/- 470) compared to vehicle (2,242 +/- 1,443). Aspirin (992 +/- 973), clopidogrel (537 +/- 483), clopidogrel plus aspirin (228 +/- 66) or PD0348292 plus aspirin (558 +/- 317) also significantly inhibited platelet deposition, although these values were not significantly different than with any dose of PD348292. Perfusion chamber platelet deposition correlated significantly with in-vivo anti-thrombotic response. In conclusion, PD0348292 inhibited arterial thrombosis comparable to aspirin plus clopidogrel. Perfusion chamber methodology may be useful in predicting in-vivo antithrombotic efficacy.