IntroductionA ruptured atherosclerotic plaque leads to exposure of deeper layers of the plaque to flowing blood and subsequently to thrombus formation. In contrast to the wealth of data on the occurrence of thrombi, little is known about the reasons why an atherosclerotic plaque is thrombogenic. One of the reasons is the relative inaccessibility of the atherosclerotic plaque. We have circumvented this problem by using 6-,gm cryostat cross sections of human coronary arteries. These sections were mounted on coverslips that were exposed to flowing blood in a rectangular perfusion chamber. In normal-appearing arteries, platelet deposition was seen on the luminal side of the intima and on the adventitia. In atherosclerotic arteries, strongly increased platelet deposition was seen on the connective tissue of specific parts of the atherosclerotic plaque. Despite the evident importance of this issue, few studies exist about the thrombogenicity of the atherosclerotic plaque. Coagulation tests with tissue extracts of atheromas (6, 7) and addition of atheroma suspension to blood in a rotating tilted closed plastic tube ("Chandler loop" model) (7-9) tended to show less thrombogenicity than expected (6,8,9). More recently histochemical studies showed that there is more tissue factor in an atherosclerotic plaque than in the normal vessel wall (10, 11). Perfusion studies with blood over hyperlipidemic rabbit atherosclerotic aortic subendothelium showed decreased platelet adhesion ( 12), whereas the injured artificially created neointima in rabbits was more thrombogenic than the injured normal intima ( 13 ). A problem of most studies is that the relevant deeper layers ofthe atherosclerotic plaque are relatively inaccessible to flowing blood. Artificial rupture of the atherosclerotic plaque in order to bring blood in contact with deeper layers has the disadvantage that it is difficult to evaluate the actual thrombogenicity because ofthe disturbed blood flow pattern. We have circumvented these problems by using cryostat cross sections of postmortem human coronary arteries. These cross sections were mounted on coverslips and anticoagulated blood was perfused over them in a rectangular perfusion chamber ( 14). These studies showed platelet deposition and thrombus formation particularly on the luminal side of the subendothelium and on the adventitia in normal vessels and strongly increased platelet deposition on the connective tissue of the atherosclerotic plaque. No platelet deposition was seen on the central lipid core of the atherosclerotic plaque.To study the cause of increased platelet deposition on the atherosclerotic plaque, perfusion studies over cross sections of atherosclerotic coronary arteries were combined with immunohistochemical studies and inhibition studies using antibodies and specific inhibitors. Methods SpecimensPostmortem coronary arteries from 25 patients with different causes of death and fresh coronary arteries from 11 patients who were undergoing cardiac transplantation were obtained from the Depart...

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

Monocyte chemoattractant protein 1 (MCP-1) has been shown to be effective for the stimulation of collateral artery formation in small and large animal models. The availability of a genetic knockout mouse enables evaluation of the importance of the role of MCP-1 in the natural course of collateral artery growth. In a total of 21 MCP-1 À=À as well as 13 of the appropriate genetic background controls (f129Sv=J X C57Bl=6JgF1), a femoral artery ligation was performed. Subsequently, a polyethylene catheter, connected to an osmotic minipump, was inserted retrogradely into the occluded femoral artery with the tip pointing upstream. Using this technique, PBS (MCP-1 À=À: n ¼ 13 and C57Bl=6J: n ¼ 13) or MCP-1 (JE; MCP-1 À=À: n ¼ 8) was delivered intra-arterially. Seven days after ligation, determination of hind limb flow was assessed by controlled tissue perfusion using differently labeled fluorescent microspheres. MCP-1 À=À mice exhibited a reduction of hind limb flow of 32.9 AE 9.2% of the unligated hind limb, compared with 55.4 AE 6.8% in C57Bl=6J mice (p < 0.01). MCP-1 À=À mice that underwent a subsequent 'rescue' treatment with MCP-1 showed a restoration of flow to a level of 47.4 AE 9.8% (p ¼ NS compared with PBS-treated C57Bl=6J). Specific immunohistochemical staining for monocytes (MOMA-2: MCP-1 À=À, n ¼ 5 and C57Bl=6J, n ¼ 5) showed a reduced number of monocytes around developing collateral arteries in the MCP-1 À=À mice. In conclusion, our data show that the absence of MCP-1 causes a strong reduction in flow restoration after femoral artery occlusion, coinciding with a reduced monocyte attraction, emphasizing the central role of this chemokine in the multifactorial process of collateral artery formation.

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Background-Arteriogenesis refers to the development of collateral conductance arteries and is orchestrated by circulating monocytes, which invade growing collateral arteries and act as suppliers of cytokines and growth factors. CD44 glycoproteins are involved in leukocyte extravasation but also in the regulation of growth factor activation, stability, and signaling. Here, we explored the role of CD44 during arteriogenesis. Methods and Results-CD44 expression increases strongly during collateral artery growth in a murine hind-limb model of arteriogenesis. This CD44 expression is of great functional importance, because arteriogenesis is severely impaired in CD44 Ϫ/Ϫ mice (wild-type, 54.5Ϯ14.9% versus CD44 Ϫ/Ϫ , 24.1Ϯ9.2%, PϽ0.001). The defective arteriogenesis is accompanied by reduced leukocyte trafficking to sites of collateral artery growth (wild-type, 29Ϯ12% versus CD44 Ϫ/Ϫ , 18Ϯ7% CD11b-positive cells/square, PϽ0.01) and reduced expression of fibroblast growth factor-2 and platelet-derived growth factor-B protein. Finally, in patients with single-vessel coronary artery disease, the maximal expression of CD44 on activated monocytes is reduced in case of impaired collateral artery formation (poor collateralization, 1764Ϯ572 versus good collateralization, 2817Ϯ1029 AU, PϽ0.05). Conclusions-For the first time, the pivotal role of CD44 during arteriogenesis is shown. The expression of CD44 increases during arteriogenesis, and the deficiency of CD44 severely impedes arteriogenesis. Maximal CD44 expression on isolated monocytes is decreased in patients with a poor collateralization compared with patients with a good collateralization.