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Biomedical subjects

James J Hathcock

Publications and source records attributed to James J Hathcock.

5 recordsLinked to original sources
PubMed2006

Flow effects on coagulation and thrombosis.

Thrombosis occurs in a dynamic rheological field that constantly changes as the thrombus grows to occlusive dimensions. In the initiation of thrombosis, flow conditions near the vessel wall regulate how quickly reactive components are delivered to the injured site and how rapidly the reaction products are disseminated. Whereas the delivery and removal of soluble coagulation factors to the vessel is thought to occur via classic convection-diffusion phenomena, the movement of cells and platelets to the injured wall is strongly augmented by flow-dependent cell-cell collisions that enhance their ability to interact with the wall. In addition, increased shear conditions have been shown to activate platelets, alter the cellular localization of proteins such as tissue factor (TF) and TF pathway inhibitor, and regulate gene production. In the absence of high shearing forces, red cells, leukocytes, and platelets can form stable aggregates with each other or cells lining the vessel wall, which, in addition to altering the biochemical makeup of the aggregate or vessel wall, effectively increases the local blood viscosity. Thus, hemodynamic forces not only regulate the predilection of specific anatomic sites to thrombosis, but they strongly influence the biochemical makeup of thrombi and the reaction pathways involved in thrombus formation.

Animals↗
PubMed2006

Phospholipid surfaces regulate the delivery of substrate to tissue factor:VIIa and the removal of product.

For many years, the essential role of tissue factor (TF) in coagulation and thrombogenesis has been recognized. The catalytic complex of TF and VIIa (TF:VIIa) is membrane-bound whereas its substrate, factor X (FX), is distributed between a phospholipid-bound fraction and one that is in true solution in 3-dimensional space. This complicates analytical solutions for the kinetic mechanisms describing this reaction because dimensionality must be preserved. We believe that, at the time of activation, FX is simultaneously bound to TF:VIIa and the phospholipid surface. The hydrolysis of a peptide bond activates FX and the product, Xa, is yet bound to the catalytic complex in a manner such that it must leave before a new molecule of X encounters the complex. This means that, in principle, the classically defined Vmax does not apply because on a surface, infinite substrate and its attendant infinite collision frequency do not apply. We show that increasing the lipid surface area available to each TF:VIIa increases the apparent k(cat) and that it approaches a maximum asymptotically, exhibiting a K(1/2) at a 40 nm lipid radius. Notably, this is of the same order as transient confinement zones that have been identified on the surface of living cells. We believe the increased lipid surface area allows the Xa to easily diffuse away from the enzyme complex along the 2D lipid surface, thereby allowing new substrate to approach the enzyme and minimizing collisions between the product and the enzyme complex (product inhibition). Thus, after Xa leaves the vicinity of the enzyme, a new FX molecule is able to bind TF:VIIa and the rate at which this complex forms cannot exceed the leaving rate of Xa from the TF:VIIa and phospholipid sites. Thus, this parameter is of critical interest. Starting with the off-rate of Xa from appropriate phospholipid surfaces, we note that the literature values differ by a factor of approximately 500. Using energy transfer techniques between 30% phosphatidylserine/70% phosphatidylcholine vesicles and human F.Xa, we measured this off rate and found it agrees closely with the Biacore generated data. We have determined the binding parameters of Xa to vesicles and a continuous supported bilayer. Our data are in excellent agreement with the data derived using a lipid coated Biacore chip.

Catalysis↗
PubMed2005

Phospholipid regulates the activation of factor X by tissue factor/factor VIIa (TF/VIIa) via substrate and product interactions.

Although the phospholipid requirement for tissue factor (TF) activity has been well-established, the mechanism by which the surface regulates enzymatic activity remains unclear. We added phospholipid vesicles to already relipidated TF (30/70 PS/PC) and found that added lipid can both enhance and inhibit the rate of factor X (F.X) activation. Using active-site-inhibited F.Xa we demonstrate that F.Xa is a more potent inhibitor of TF/VIIa at lower lipid concentrations, and that this inhibition is attributable to high surface occupancy by F.Xa near the enzyme. We also find that exactly twice as many F.Xa molecules are bound to a lipid surface at saturation as F.X, and that a dimer model of F.Xa binding to the lipid can account for the experimentally observed, preferential binding of F.Xa (compared to F.X) to phospholipid surfaces. We manipulated the amount of phospholipid available to each TF molecule by controlling vesicle size and the number of TF molecules per vesicle and found that, as the 2D radius of phospholipid available to each TF molecule was increased, the observed k(cat) increased hyperbolically toward a maximum or "true k(cat)". At a 2D lipid radius of approximately 37 nm, the observed k(cat) was 50% of the "true k(cat)". Thus, phospholipid surface serves as a conduit for F.X presentation and F.Xa removal, and the rate at which F.Xa leaves the vicinity of the enzyme, either by lateral diffusion or desorption from the surface, regulates the rate of F.X activation. We argue that these findings require reevaluation of existing models of coagulation.

Binding, Competitive↗
PubMed2004

Platelet deposition inhibits tissue factor activity: in vitro clots are impermeable to factor Xa.

Upon plaque rupture or vascular injury, tissue factor (TF) protein in the vessel wall becomes exposed to flowing blood, initiating a cascade of reactions resulting in the deposition of fibrin and platelets on the injured site. Paradoxically, the growing thrombus may act as a barrier, restricting the convective and diffusive exchange of substrates and coagulation products between the blood and reactive vessel wall, thus limiting the role TF plays in thrombus growth. In this study, various in vitro, platelet-fibrin clots were prepared on TF:VIIa-coated surfaces and the rate at which factor (F) X in the well-mixed clot supernatant permeates the clot and is converted to X(a) was monitored over several hours. The apparent diffusion coefficients of FX((a)) in fibrin and platelet-fibrin clots at 37 degrees C was 2.3 x 10(-7) and 5.3 x 10(-10) cm(2)/second, respectively, indicating that the mean time required for FX((a)), and likely FIX((a)), to diffuse 1 mm in a fibrin clot is 4 hours, and in the presence of platelets, 3.6 months. As complete human thrombotic occlusion has been observed within 10 minutes, an alternative source of procoagulant activity that can localize to the outer surface of growing thrombi, such as platelet factor XI or blood-borne TF, appears essential for rapid thrombus growth.

Blood Coagulation↗