Mann, Kenneth G.

Background It has been observed that trauma patients have elevated plasma procoagulant activity that could be assigned to an elevated concentration of tissue factor (TF). However, in many instances there is a discrepancy between the levels of TF and the procoagulant activity observed. We hypothesized that factor XIa (FXIa) could be responsible for this additional activity and that the presence and levels of both proteins could correlate with trauma severity. Methods Citrate plasma from 98 trauma patients (47 blunt, 17 penetrating, and 34 thermal) were evaluated in clotting assays for the presence of FXIa and TF activity using respective inhibitory antibodies. Results When the three trauma patient groups were divided into two cohorts (Injury Severity Score [ISS] > 25 and ISS ≤ 25), higher frequencies and concentrations of both TF and FXIa were observed for all the more severe injury subgroups. Conclusions The majority of trauma patients have active FXIa in their plasma, with a significant fraction having active TF as well. Additionally, both TF and FXIa frequency and concentration directly relate to trauma severity. These data suggest the use of these two proteins as potential markers for the stratification of trauma patients.

SummaryWhen 125I-labeled canine prothrombin was given to normal adult dogs intravenously, it was calculated that 240% of the plasma prothrombin crossed the capillary barrier per day, 410% of the interstitial prothrombin returned to the blood stream daily, and 79% of the plasmatic prothrombin was catabolized per day. These data are in close agreement with those observed for bovine prothrombin in calves by Takeda (1970).When derived from normal dog prothrombin, prethrombin-1 is a mixture of 2 polypeptides, one larger than the other, and both present in about equal amounts. The longer peptide, “prethrombin-1-long,” was catabolized twice as fast as prothrombin, and the shorter, “prethrombin-1-short,” 4 times faster. Prothrombin fragment-1 was catabolized by the normal dog still more rapidly.The catabolism of prothrombin was not accelerated in 3 dogs receiving continuous infusions of a thromboplastic emulsion of dog brain. Nor was the level of prothrombin in their plasma remarkably altered.

Highly purified human Factor V was used for the development of a competitive double antibody radioimmunoassay (RIA) using 125I-human Factor V, burro anti-human Factor V antisera as the primary antibody and goat anti-burro antisera as the precipitating antibody. The standard curve allows the detection of as little as 20 ng of Factor V per ml of plasma. With this specific RIA for human Factor V, we have measured the level of Factor V in the plasma and platelets of normal individuals. The normal level of Factor V in plasma ranges from 4 to 14 μg per ml, with the average value equal to 7.0 ± 2.0 μg/ml (n = 64; 33 females; 31 males; 22 to 61 years of age). There appeared to be no correlation between antigen levels and age or sex. Factor V clotting assays were consistent with the RIA data for any given plasma preparation providing freshly drawn plasma was used in the bioassay. The bioassay data were quantitated based upon the specific activity of purified plasma Factor V; 1.7 units of Factor V equals 1 μg of protein. Plasma Factor V antigen levels were not affected by lyophilization of the plasma, prolonged storage of the plasma at -20°C or intentional conversion of the plasma to serum. The levels of Factor V present in washed human platelets were also determined using the RIA. Assay of washed platelets lysed in 0.2% Triton X-100 indicated that 0.6 to 0.85 μg of Factor V was present per 2.5 × 108 platelets (4400 to 6200 molecules of Factor V per platelet). This result is in marked contrast to our observations for the bovine system, where we found that bovine platelets possess approximately 400 to 800 molecules of Factor V per platelet, and plasma Factor V levels range from 30 to 50 μg per ml. In the bovine system, the platelets possess approximately 1% of the total Factor V present, while in human blood, the platelets possess as much as 10 to 15% of the total Factor V present.

Abstract We have compared two assays for osteocalcin (BGP, bone gla protein), one based on a monoclonal antibody, the other based on a polyclonal serum, in an effort to better understand the factors that contribute to the wide variation reported for osteocalcin normal ranges. The two assays compared well using serum samples, with a correlation coefficient of 0.9071 (n = 48). However, the monoclonal antibody assay returns values increased over the polyclonal assay by approximately 1.5-fold. Gel permeation studies indicate that these larger values are due primarily to increased reactivity in the monoclonal assay with high- and low-molecular-weight forms of osteocalcin in plasma; both assays give similar reactions to intact osteocalcin. Analysis of samples from individuals with increased bone resorption due to parathyroid hormone administration reveals that the decrease seen in osteocalcin values after hormone infusion occurs primarily in the fraction that corresponds to intact osteocalcin. During the course of these studies, we re-evaluated the extinction coefficient for osteocalcin, arriving at the value E = 1.33 mg/ml−1, cm−1. We also observed a significant negative interference in both assays (and a commercial assay) caused by hemolysis. This interference is due to proteolysis of osteocalcin by enzymatic activity released from the lysed red cells.

Abstract Serum bone Gla-prolein (BGP), also called osteocalcin, is a specific and sensitive measure of bone turnover in a variety of metabolic bone disorders. Although some BGP diffuses into the circulation after synthesis by osteoblasts, most is incorporated into bone matrix where it remains until bone is resorbed. Thus, serum BGP could reflect bone formation, bone resorption, or a combination of both. The relationship of serum BGP to the components of bone turnover was evaluated in 18 normal women (mean age 48 yr; range 30-70) who received a continuous 24-h intravenous infusion of the 1-34 synthetic fragment of bovine parathyroid hormone. Mean ± SE for urinary hydroxyproline excretion, an index of bone resorption, increased (from 22.7 ± 2.2 to 38.5 ± 3.7 μg/100 ml glomerular filtrate [GF], p < .001), whereas levels of serum alkaline phosphatase, an index of bone formation, were unchanged (from 20 ± 1 to 20 ± 1 U/liter, NS). Despite the increase in bone resorption, levels of serum BGP decreased (from 8.8 ± 0.8 to 6.8 ng/dl, p < .001). The data suggest that circulating levels of BGP are a measure of bone formation but, at least in subjects with normal renal function, not a measure of bone resorption. Presumably BGP in bone matrix is degraded during osteoclastic resorption into fragments that either are not recognized by an antiserum raised against the native molecule or are rapidly cleared from the circulation.

In seven strains of cultured normal human osteoblast-like cells, a mean of 1615 molecules of tritium-labeled 17β-estradiol per cell nucleus could be bound to specific nuclear sites. The nuclear binding of the labeled steroid was temperature-dependent, steroid-specific, saturable, and cell type-specific. These are characteristics of biologically active estrogen receptors. Pretreatment with 10 nanomolar estradiol in vitro increased the specific nuclear binding of progesterone in four of six cell strains, indicating an induction of functional progesterone receptors. RNA blot analysis demonstrated the presence of messenger RNA for the human estrogen receptor. The data suggest that estrogen acts directly on human bone cells through a classical estrogen receptor-mediated mechanism.

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.