Background: COVID-19 is a major pandemic that has killed more than 196,000 people. The COVID-19 disease course is strikingly divergent. Approximately 80-85% of patients experience mild or no symptoms, while the remainder develop severe disease. The mechanisms underlying these divergent outcomes are unclear. Emerging health disparities data regarding African American and homeless populations suggest that vitamin D insufficiency (VDI) may be an underlying driver of COVID-19 severity. To better define the VDI-COVID-19 link, we determined the prevalence of VDI among our COVID-19 intensive care unit (ICU) patients. Methods: In an Institutional Review Board approved study performed at a single, tertiary care academic medical center, the medical records of COVID-19 patients were retrospectively reviewed. Subjects were included for whom serum 25-hydroxycholecalcifoerol (25OHD) levels were determined. COVID-19-relevant data were compiled and analyzed. We determined the frequency of VDI among COVID-19 patients to evaluate the likelihood of a VDI-COVID-19 relationship. Results: Twenty COVID-19 patients with serum 25OHD levels were identified; 65.0% required ICU admission.The VDI prevalence in ICU patients was 84.6%, vs. 57.1% in floor patients. Strikingly, 100% of ICU patients less than 75 years old had VDI. Coagulopathy was present in 62.5% of ICU COVID-19 patients, and 92.3% were lymphocytopenic. Conclusions: VDI is highly prevalent in severe COVID-19 patients. VDI and severe COVID-19 share numerous associations including hypertension, obesity, male sex, advanced age, concentration in northern climates, coagulopathy, and immune dysfunction. Thus, we suggest that prospective, randomized controlled studies of VDI in COVID-19 patients are warranted.

Activation of prothrombin by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X(a) of human prothrombin was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-1) s(-1), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X(a), (2) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X(a) for rapid conversion to thrombin.

Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor X a by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482-7491). In the present study, we locate three C6PS binding sites to different domains of factor X a using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor X a . Our results demonstrate that the structural responses of human and bovine factor X a to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor X a are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the ␥-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca 2؉ (k d ϳ 1 mM), although this PS site does not influence the functional response of factor X a . Second, a Ca 2؉ -dependent binding site is in the epidermal growth factor domains (EGF NC ) that are linked by Ca 2؉ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X a . Third, a Ca 2؉ -requiring site seems to be in the EGF C -catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGF NC , and catalytic domains in the presence of Ca 2؉ , meaning that PS regulation of factor X a involves linkage between widely separated parts of the protein.The substantial effects of soluble phosphatidylserine (C6PS 1 ) on the kinetics of prothrombin activation by factor X a(1) and on the structure of factor X a , as documented here, indicate that phosphatidylserine (PS) may act as an allosteric regulator of prothrombin activation. PS located on the cytoplasmic face of resting platelet plasma membranes is exposed on the surface of activated platelet vesicles (2, 3). The implication of this PS exposure and of the effect of PS on factor X a and on its ability to catalyze activation of prothrombin is that PS may act as a second messenger in regulating thrombin formation. Because of the crucial role of thrombin in hemostasis, the exposure of PS may be a crucial regulatory step in blood coagulation. To better define this regulatory process, it is important to know the locations of the PS binding sites on factor X a . The organization of factor X into structural domains is illustrated below in Fig. 1. Factor X consists of two peptides. The light chain consists of an N terminus ␥-carboxyglutamic acidrich region (Gla module) and two Cys-rich cassette modules. The heavy chain consists of the serine protease catalytic domain. The two cassette modules of the light chain show strong sequence and structural homology to epidermal growth factor (EGF) (4) and are thus referred to as EGF N and EGF C , where N and C indicate the domain nearer to the N and C termini, respectively. Crystal structures o...

Factor X a (FX a ) binding to factor V a (FV a ) on plateletderived membranes containing surface-exposed phosphatidylserine (PS) forms the "prothrombinase complex" that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FV a , FV a1 and FV a2 . These two isoforms differ by a 3-kDa polysaccharide chain ( ). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.The final step in the blood coagulation cascade involves the activation of prothrombin to thrombin, which is the central enzyme of the coagulation system. This activation requires assembly of an enzyme complex, called prothrombinase (1), which consists of blood coagulation factors X a (a serine protease) and V a (a cofactor), Ca 2ϩ , and membranous vesicles derived from stimulated platelets (2). Several studies (3-5) have suggested that phosphatidylserine (PS) 1 might play a specific role in prothrombin activation. PS is asymmetrically distributed to the cytoplasmic surface of resting platelet membranes (6) but is exposed when human platelets are activated (7). It has become clear only very recently that PS regulates the structure and function of factors X a and V a (8, 10). 2 Here we explore further the extent of this regulation.Factor V exists in plasma as an inactive, single chain glycoprotein with a molecular mass of 330 kDa. The active form of factor V, FV a , has a central domain removed to yield a heterodimer composed of two chains, a heavy chain (M r ϭ 94,000 in the bovine species; 105,000 in human) and a heterogeneous light chain (M r ϭ 74,000 in FV a1 or 71,000 in FV a2 ). The heavy and light chains form a tight complex in the presence of a calcium ion (11). The heterogeneity in the light chain is seen in both the bovine and human molecules. In the human form, it appears to arise from glycosylation of Asn 2181 at the C-terminal end of the light chain (12). Prothrombinase complexes assembled from the two molecular species derived from human plasma are observed to have somewhat different cofactor activities (13,14). This has been attributed to substantially different affinities for binding to membranes (13). However, our lab has reported that the two forms of both bovine and human FV a bind to membranes with only ϳ3-fold different affinities (12,14). This suggests that differences in the ability to support prothrombinase activity must reflect either different binding between factors FX a and FV a1 versus FV a2 or different intrinsic activities of the FX a ⅐FV a1 and FX a ⅐FV a2 complexes. Although our results have favored the former possibility (14), it has been difficult to prove this unambiguously because it is difficult to measure precisely the interaction between FX a and FV a on a membrane surface (15).The presence of FV a in a reaction mixture is critical to obtaining a maximal and physiologicall...

Activation of prothrombin to thrombin is catalyzed by a "prothrombinase" complex, traditionally viewed as factor X(a) (FX(a)) in complex with factor V(a) (FV(a)) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through "channeling" (activation without the release of an intermediate). We ask here whether the PS molecule itself and not the membrane surface is sufficient to produce the fully active human "prothrombinase" complex in solution. Both FX(a) and FV(a) bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FX(a) and FV(a2) (light isoform of FV(a)), these proteins form a tight (Kd = 0.6 +/- 0.09 nM at 37 degrees C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels shows that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FX(a) and one FV(a2), further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2 x 10(8) M(-1) s(-1)) and via the same reaction pathway (50-60% channeling, with the rest via the meizothrombin intermediate) as the activation catalyzed by a complex assembled on PS-containing membranes (4.4 x 10(8) M(-1) s(-1)). These results question the accepted role of PS membranes as providing "dimensionality reduction" and favor a regulatory role for platelet-membrane-exposed PS.

Previous work has shown that two molecules of a soluble form of phosphatidylserine, C6PS, bind to human and bovine factor X(a). Activity measurements along with the fluorescence of active-site-labeled human factor X(a) showed that two linked sites specifically regulate the active site conformation and proteolytic activity of the human enzyme. These results imply, but cannot demonstrate, a C6PS-induced factor X(a) conformational change. The purpose of this paper is to extend these observations to bovine factor X(a) and to demonstrate that they do reflect conformational changes. We report that the fluorescence of active-site-labeled bovine factor X(a) also varied with C6PS concentration in a sigmoidal manner, whereas amidolytic activity of unlabeled enzyme varied in a simple hyperbolic fashion, also as seen for human factor X(a). C6PS induced a 70-fold increase in bovine factor X(a)'s autolytic activity, consistent with the 60-fold increase in proteolytic activity reported for human factor X(a). In addition, circular dichroism spectroscopy clearly demonstrated that C6PS binding to bovine factor X(a) induces secondary structural changes. In addition, C6PS binding to the tighter of the two sites triggered structural changes that lead to Ca(2+)-dependent dimer formation, as demonstrated by changes in intrinsic fluorescence and quantitative native gel electrophoresis. Dimerization produced further change in secondary structure, either inter- or intramolecularly. These results, along with results presented previously, support a model in which C6PS binds in a roughly sequential fashion to two linked sites whose occupancy in both human and bovine factor X(a) elicits different structural and functional responses.

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).

Tightly associated factor V(a) (FVa) and factor X(a) (FXa) serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. We have previously shown that (1) a soluble form of PS (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 domain. We expressed human factor V(a) (rFVa) with mutations in either the C1 domain (Y1956,L1957)A, the C2 domain (W2063,W2064)A, or both C domains (Y1956,L1957,W2063,W2064)A. Mutations in the C1 and C1-C2 domains of rFVa reduced the rate of activation of prothrombin to thrombin by FXa in the presence of 400 muM C6PS by 14 000- to 15 000-fold relative to either wild-type or C2 mutant factor rFVa. The K(d')s of FXa binding with rFVa (wild-type, C2 mutant, C1 mutant, and C1-C2 mutant) were 3, 4, 564, and 624 nM, respectively. Equilibrium dialysis experiments detected binding of 4, 3, and 2 molecules of C6PS to wild-type rFVa, C1-mutated, and C1,C2-mutated rFVa, respectively. Because FVa heavy chain binds 2 molecules of C6PS, we conclude that both C2 and C1 domains bind one C6PS, with binding to the C1 domain regulating prothrombinase complex assembly.