Population dynamics of the long-snouted seahorse (Hippocampus guttulatus Cuvier, 1829) in the Mar Menor coastal lagoon.

A 10-year monitoring program was developed to quantify the population dynamics of the long-snouted seahorse population in the Mar Menor coastal lagoon. Based on 985 underwater visual censuses, we estimated the long-snouted seahorse (Hippocampus guttulatus Cuvier, 1829) population size in the Mar Menor lagoon and its reduction in size in the last decades, as well as the effect of eutrophication crises in 2016 and 2019 on the species. The annual recruitment for the 2013-2020 period was estimated by comparing the relative abundance of early seahorse life stages in the ichthyoplankton. The density ranged from 0.0458 specimens/m3 at the beginning of the sampling period to 0.0004 at the end, showing a statistically significant difference between the three analyzed periods (Hgl=2 = 14.0, p = 0.001). The long-snouted seahorse population from the Mar Menor lagoon exemplifies the impact of fishing activities and human pressure, especially euxinic episodes and habitat destruction. As a result of this, the Mar Menor population has decreased from several million specimens to a few thousand, in only three decades. This species showed considerable resilience, the seahorse population began to recover once fishing activity stopped. In contrast, the long-snouted seahorse showed high vulnerability to habitat loss and an episodic flooding event. Adult seahorses showed preferences for highly complex habitats, especially Caulerpa prolifera-Cymodocea nodosa mixed meadows and habitats of high complexity and anthropogenic origin, such as harbors, jetties, or breakwaters. In contrast, juvenile seahorses preferred monotonous seabeds with low complexity, such as the sandy beds that are characteristic of the Mar Menor lagoon littoral.

The ATP-sensitive K+ channel mediates hypotension in endotoxemia and hypoxic lactic acidosis in dog.

Endotoxemia causes hypotension characterized by vasodilation and resistance to vasopressor agents. The molecular mechanisms responsible for these changes are unclear. The ATP-regulated K+ (K+ATP) channel has recently been found to be an important modulator of vascular smooth muscle tone which may transduce local metabolic changes into alterations of vascular flow. We report here that in endotoxic hypotension, the sulfonylurea glyburide, a specific inhibitor for the K+ATP channel, caused vasoconstriction and restoration of blood pressure. Glyburide also induced vasoconstriction and restoration of blood pressure in the vasodilatory hypotension caused by hypoxic lactic acidosis, while it was ineffective in the hypotension induced by sodium nitroprusside. Thus, vasodilation and hypotension in septic shock are, at least in part, due to activation of the K+ATP channel in vascular smooth muscle, and anaerobic metabolism with acidosis is a sufficient stimulus for channel activation. Because anaerobic metabolism and acidosis are common features in shock of any etiology, sulfonylureas may be effective therapeutic agents in the treatment of shock.

Local generation of angiotensin II as a mechanism of regulation of peripheral vascular tone in the rat.

Renin is present in vascular smooth muscle cells and has been shown to coexist with angiotensins I (AI) and II (AII) in many cell types. Accordingly, we postulated that the renin-angiotensin system controls vascular tone, not by the action of circulating renal renin but rather, by the local generation of angiotensin by vascular renin. Isolated rat hindquarters were perfused in vitro with Krebs-Henseleit buffer containing 7% albumin, and flow-adjusted to obtain a perfusion pressure of approximately 90 mmHg. Infusion of 4.8 nmol X min-1 for 5 min of AII or AI markedly increased perfusion pressure. An identical dose of the synthetic tetradecaptide of renin substrate (TDCP-RS) increased pressure similarly to AI. The pressure increase evoked by TDCP-RS was markedly decreased by captopril and by two different peptides that inhibit renin. Renin activity in the perfusate, incubated with semipurified rat renin substrate, was 21 +/- 3 pg AI X ml-1 X h-1 (mean +/- SEM) at 15 min of perfusion and 47 +/- 4 pg AI X ml-1 X h-1 at 45 min (n = 9; P less than 0.01). When TDCP-RS was infused at 4.8 nmol X min-1 for 5 min in the presence of captopril, AI in the perfusate increased linearly at a rate of 16.5 pmol X min-1 for 10 min (n = 5). The results indicate that TDCP-RS constricted the vasculature by its conversion to AII and suggest that AII was generated from a two-step hydrolysis of TDCP-RS by renin and converting enzyme. The data thus suggest that the renin-angiotensin system controls vascular tone by the local generation of AII by renin and converting enzyme in the vasculature.

An endothelial growth factor involved in rat renal development.

In the kidney, there is a close and intricate association between epithelial and endothelial cells, suggesting that a complex reciprocal interaction may exist between these two cell types during renal ontogeny. Thus, we examined whether metanephrogenic mesenchymal cells secrete endothelial mitogens. With an endothelial mitogenic assay and sequential chromatography of the proteins in the media conditioned by a cell line of rat metanephrogenic mesenchymal cells (7.1.1 cells), we isolated a protein whose amino acid analysis identified it as hepatoma-derived growth factor (HDGF). Media conditioned with Cos-7 cell transfected with HDGF cDNA stimulated endothelial DNA synthesis. With immunoaffinity purified antipeptide antibodies, we found that HDGF was widely distributed in the renal anlage at early stages of development but soon concentrated at sites of active morphogenesis and, except for some renal tubules, disappeared from the adult kidney. From a 7.1.1 cells cDNA library, a clone of most of the translatable region of HDGF was obtained and used to synthesize digoxigenin-labeled riboprobes. In situ hybridization showed that during kidney development mRNA for HDGF was most abundant at sites of nephron morphogenesis and in ureteric bud cells while in the adult kidney transcripts disappeared except for a small population of distal tubules. Thus, HDGF is an endothelial mitogen that is present in embryonic kidney, and its expression is synchronous with nephrogenesis.

The effect of altered sodium balance upon renal vascular reactivity to angiotensin II and norepinephrine in the dog. Mechanism of variation in angiotensin responses.

The mechanism whereby the vasoconstrictor response to angiotensin II (AII) is influenced by sodium balance or disease is unclear. To explore this question, the renal vascular responses (RVR) to intrarenal injections of subpressor doses of AII and norepinephrine were studied in dogs with an electromagnetic flowmeter. Acute and chronic sodium depletion increased plasma renin activity (PRA) and blunted the RVR to AII, while acute sodium repletion and chronic sodium excess plus desoxycorticosterone acetate decreased PRA and enhanced the RVR to AII. The magnitude of the RVR to AII was inversely related to PRA. The RVR to norepinephrine was unaffected by sodium balance and was not related to PRA. Inhibition of the conversion of angiotensin I to AII by SQ 20,881 during sodium depletion lowered mean arterial blood pressure (MABP), increased renal blood flow (RBF), and enhanced the RVR to AII but not to norepinephrine. Administration of bradykinin to chronically sodium-depleted dogs also lowered the MABP and increased RBF but had no effect on the RVR to AII. SQ 20,881 had no effect on MABP, RBF, or the RVR to AII in the dogs with chronic sodium excess and desoxycorticosterone acetate. Administration of indomethacin to chronically sodium-depleted dogs lowered RBF but did not influence the RVR to AII. The results indicate that the RVR to AII is selectively influenced by sodium balance and that the magnitude of the response is inversely related to the availability of endogenous AII. The data did not suggest that the variations in the RVR to AII were because of direct effects of sodium on vascular contraction, changes in the number of vascular AII receptors, or the renal prostaglandins. The results are consistent with the hypothesis that the vasoconstrictor effect of AII in the renal vasculature is primarily dependent upon the degree to which the AII vascular receptors are occupied by endogenous hormone.

Energy dependence of urinary bicarbonate secretion in turtle bladder.

Addition of HCO3- to the serosal side (S) of the isolated turtle bladder results in a HCO3- flow from S to the mucosal side (M) which markedly reduces the net rate of acid secretion. To characterize the driving forces for this downhill HCO3- flow, the effects of metabolic inhibitors and substrates were examined. In short-circuited bladders with the M pH lowered to the point of zero net H+ secretion, the rate of HCO3- entry into M in response to a 20-mM HCO3- gradient was measured by pH stat titration. Deoxygenation reduced the HCO3- flux from 1.24 plus or minus 0.1 mum/h/8 cm2 (SEM) to 0.50 plus or minus 0.1 muM/h with glucose (2 times 10-3 M) AND FROM 1.32 PLUS OR MINUS TO 0.47 PLUS OR MINUS 0.1 MUM/h without glucose. A similar reduction (61 per cent) was observed in the presence of 1 per cent C92. Dinitrophenol (10-4 M), cyanide (10-3 M), and deoxyglucose (10-2 M) inhibited the HCO3- flux by 39 per cent, 37 per cent, and 38 per cent, respectively. The combination of any of these inhibitors with N2 caused the same inhibition as N2 alone. In bladders depleted of substrate, pyruvate (5 times 10-3 M) increased the HCO3- flux from 0.36 plus or minus 0.05 to 0.58 plus or minus 0.01 muM/h (P smaller than 0.005); the increment was abolished by deoxygenation. The results indicate that the bulk of the downhill HCO3- flow in this system is dependent on metabolic energy derived primarily from oxidative sources, and that this energy-dependent flow approximates the electroneutral component of HCO3- secretion that is coupled to Cl- absorption.

Increased renal secretion of norepinephrine and prostaglandin E2 during sodium depletion in the dog.

To determine whether vasoactive renal hormones modulate renal blood flow during alterations of sodium balance, simultaneous measurements of arterial and renal venous concentrations of norepinephrine and prostaglandin E2 (PGE2) and of plasma renin activity, as well as renal blood flow and systemic hemodynamics were carried out in 24 sodium-depleted and 28 sodium-replete anesthetized dogs. The mean arterial blood pressure of the sodium depleted dogs was not significantly different from that of the animals fed a normal sodium diet, but cardiac output was significantly lower (3.07 +/- 0.18 vs. 3.77 +/- 0.17 liters/min, mean +/- SEM; P < 0.01). Despite the higher total peripheral vascular resistance in the sodium-depleted dogs (46.1 +/- 2.9 vs. 37.0 +/- 2.1 arbitrary resistance U; P < 0.02), the renal blood flow and renal vascular resistance were not significantly different in the two groups. The arterial plasma renin activity and concentration of norepinephrine were higher in the sodium-depleted animals than in the controls; the arterial concentration of PGE2 was equal in both groups. The renal venous plasma renin activity was higher in the sodium-depleted dogs. Similarly, the renal venous norepinephrine concentration was higher in the sodium-depleted dogs than in the controls (457 +/- 44 vs. 196 +/- 25 pg/ml; P < 0.01); renal venous PGE2 concentration was also higher in the sodium depleted dogs (92 +/- 22 vs. 48 +/- 11 pg/ml; P < 0.01). Administration of indomethacin to five sodium-replete dogs had no effect on renal blood flow. In five sodium-depleted dogs indomethacin lowered renal blood flow from 243 +/- 19 to 189 +/- 30 ml/min (P < 0.05) and PGE2 in renal venous blood from 71 +/- 14 to 15 +/- 2 pg/ml (P < 0.02). The results indicate that moderate chronic sodium depletion, in addition to enhancing the activity of the renin-angiotensin system, also increases the activity of the renal adrenergic nervous system and increases renal PGE2 synthesis. In sodium-depleted dogs, inhibition of prostaglandin synthesis was associated with a significant decrease in renal blood flow. The results suggest that the renal blood flow is maintained during moderate sodium depletion by an effect of the prostaglandins to oppose the vasoconstrictor effects of angiotensin II and the renal sympathetic nervous system.

Participation of the prostaglandins in the control of renal blood flow during acute reduction of cardiac output in the dog.

To determine whether renal prostaglandins participate in the regulation of renal blood flow during acute reduction of cardiac output, cardiac venous return was decreased in 17 anesthetized dogs by inflating a balloon placed in the thoracic inferior vena cava. This maneuver decreased cardiac output from 3.69+/-0.09 liters/min (mean+/-SEM) to 2.15+/-0.19 liters/min (P < 0.01) and the mean arterial blood pressure from 132+/-4 to 111+/-5 mm Hg (P < 0.01) and increased total peripheral vascular resistance from 37.6+/-2.5 to 57.9+/-4.8 arbitrary resistance units (RU) (P < 0.01). In marked contrast, only slight and insignificant decreases in the renal blood flow from 224+/-16 to 203+/-19 ml/min and renal vascular resistance from 0.66+/-0.06 to 0.61+/-0.05 arbitrary resistance units (ru) were observed during inflation of the balloon. Concomitant with these hemodynamic changes, plasma renin activity and plasma norepinephrine concentration increased significantly in both the arterial and renal venous bloods. Plasma concentration of prostaglandin E(2) in renal venous blood increased from 34+/-6 to 129+/-24 pg/ml (P < 0.01). The subsequent administration of indomethacin or meclofenamate had no significant effect on mean arterial pressure, cardiac output, and total peripheral vascular resistance, but reduced renal blood flow from 203+/-19 to 156+/-21 ml/min (P < 0.01) and increased renal vascular resistance from 0.61+/-0.05 to 1.05+/-0.21 ru (P < 0.01). Simultaneously, the plasma concentration of prostaglandin E(2) in renal venous blood fell from 129+/-24 to 19+/-3 pg/ml (P < 0.01). Administration of indomethacin to five dogs without prior obstruction of the inferior vena cava had no effect upon renal blood flow or renal vascular resistance. The results indicate that acute reduction of cardiac output enhances renal renin secretion and the activity of the renal adrenergic nerves as well as renal prostaglandin synthesis without significantly changing renal blood flow or renal vascular resistance. Inhibition of prostaglandin synthesis during acute reduction of cardiac output results in an increased renal vascular resistance and reduced renal blood flow. Accordingly, that data provide evidence that renal prostaglandins counteract in the kidney the vasoconstrictor mechanisms activated during acute reduction of cardiac output.

Tissue inhibitor of metalloproteinase-2 stimulates mesenchymal growth and regulates epithelial branching during morphogenesis of the rat metanephros.

Development of the embryonic kidney results from reciprocal signaling between the ureteric bud and the metanephric mesenchyme. To identify the signaling molecules, we developed an assay in which metanephric mesenchymes are rescued from apoptosis by factors secreted from ureteric bud cells (UB cells). Purification and sequencing of one such factor identified the tissue inhibitor of metalloproteinase-2 (TIMP-2) as a metanephric mesenchymal growth factor. Growth activity was unlikely due to TIMP-2 inhibition of matrix metalloproteinases because ilomastat, a synthetic inhibitor of these enzymes, had no mesenchymal growth action. TIMP-2 was also involved in morphogenesis of the ureteric bud, inhibiting its branching and changing the deposition of its basement membrane; these effects were due to TIMP-2 inhibition of matrix metalloproteinases, as they were reproduced by ilomastat. Thus, TIMP-2 regulates kidney development by at least 2 distinct mechanisms. In addition, TIMP-2 was secreted from UB cells by mesenchymal factors that are essential for ureteric bud development. Hence, the mesenchyme synchronizes its own growth with ureteric morphogenesis by stimulating the secretion of TIMP-2 from the ureteric bud.

Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2.

Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.

Gonioscopy in the dog: inter-examiner variability and the search for a grading scheme.

OBJECTIVES: To investigate inter-examiner variability in gonioscopic evaluation of pectinate ligament abnormality in dogs and to assess level of inter-examiner agreement for four different gonioscopy grading schemes. MATERIALS AND METHODS: Two examiners performed gonioscopy in 98 eyes of 49 Welsh springer spaniel dogs and estimated the percentage circumference of iridocorneal angle affected by pectinate ligament abnormality to the nearest 5%. Percentage scores assigned to each eye by the two examiners were compared. Inter-examiner agreement was assessed following assignment of the percentage scores to each of four grading schemes by Cohen's kappa statistic. RESULTS: There was a strong positive correlation between the results of the two examiners (R=0·91). In general, Examiner 1 scored individual eyes higher than Examiner 2, especially for eyes in which both examiners diagnosed pectinate ligament abnormality. A "good" level of agreement could only be achieved with a gonioscopy grading scheme of no more than three categories and with a relatively large intermediate bandwidth (κ=0·68). CLINICAL SIGNIFICANCE: A three-tiered grading scheme might represent an improvement on hereditary eye disease schemes which simply classify dogs to be either "affected" or "unaffected" for pectinate ligament abnormality. However, the large intermediate bandwidth of this scheme would only allow for the additional detection of those dogs with marked progression of pectinate ligament abnormality which would be considered most at risk of primary closed-angle glaucoma.

Prevalence and progression of pectinate ligament dysplasia in the Welsh springer spaniel.

OBJECTIVES: To determine the prevalence of pectinate ligament dysplasia in a large group of Welsh springer spaniels; to investigate associations between pectinate ligament dysplasia and age, sex and intraocular pressure and between intraocular pressure and age and sex; and to investigate progression of pectinate ligament dysplasia in individual dogs. METHODS: In a prospective study, gonioscopy was performed in both eyes of 227 Welsh springer spaniels and intraocular pressure measured by rebound tonometry. Eyes were classified as "unaffected" if 0% of the iridocorneal angle was affected with pectinate ligament dysplasia (grade 0), "mildly affected" if <20% was affected (grade 1), "moderately affected" if 20 to 90% was affected (grade 2) and "severely affected" if >90% was affected (grade 3). In a retrospective study, progression of pectinate ligament dysplasia over time was investigated for 65 dogs. RESULTS: One hundred and thirty-nine of 227 dogs (61·2%) were affected by pectinate ligament dysplasia (grades 1 to 3) and 82/227 (36·2%) were moderately or severely affected. There was a significant association between pectinate ligament dysplasia and age. There were no associations between pectinate ligament dysplasia and intraocular pressure or pectinate ligament dysplasia and sex. Thirty-five of 65 dogs (53·8%) demonstrated progression of pectinate ligament dysplasia. CLINICAL SIGNIFICANCE: Prevalence of pectinate ligament dysplasia was high despite widespread screening and selection against the condition. Our data indicate that gonioscopic features of pectinate ligament dysplasia can progress in the Welsh springer spaniel. Dogs deemed unaffected at an early age may subsequently be diagnosed with pectinate ligament dysplasia.

Integrated Microfluidic Aptasensor for Mass Spectrometric Detection of Vasopressin in Human Plasma Ultrafiltrate.

We present a microfluidic aptamer-based biosensor for detection of low-molecular-weight biomarkers in patient samples. Using a microfluidic device that integrates aptamer-based specific analyte extraction, isocratic elution, and detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we demonstrate rapid, sensitive and label-free detection of arginine vasopressin (AVP) in human plasma ultrafiltrate. AVP molecules in complex matrices are specifically captured by an aptamer that is immobilized on microbeads via affinity binding in a microchamber. After the removal of unbound, contaminating molecules through washing, aptamer-AVP complexes are thermally disrupted via on-chip temperature control. Released AVP molecules are eluted with purified water and transferred to a separate microchamber, and deposited onto a single spot on a MALDI plate via repeated, piezoelectrically actuated ejection, which enriches AVP molecules over the spot area. This integrated on-chip sample processing enables the quantitative detection of low-abundance AVP by MALDI-TOF mass spectrometry in a rapid and label-free manner. Our experimental results show the detection of AVP in human plasma ultrafiltrate as low as physiologically relevant picomolar concentrations via aptamer-based selective preconcentration, demonstrating the potential of our approach as a rapid (~ 1hr), sensitive clinical AVP assay.

Reversal by vasopressin of intractable hypotension in the late phase of hemorrhagic shock.

BACKGROUND: Hypovolemic shock of marked severity and duration may progress to cardiovascular collapse unresponsive to volume replacement and drug intervention. On the basis of clinical observations, we investigated the action of vasopressin in an animal model of this condition. METHODS AND RESULTS: In 7 dogs, prolonged hemorrhagic shock (mean arterial pressure [MAP] of approximately 40 mm Hg) was induced by exsanguination into a reservoir. After approximately 30 minutes, progressive reinfusion was needed to maintain MAP at approximately 40 mm Hg, and by approximately 1 hour, despite complete restoration of blood volume, the administration of norepinephrine approximately 3 micrograms . kg(-1). min(-1) was required to maintain this pressure. At this moment, administration of vasopressin 1 to 4 mU. kg(-1). min(-1) increased MAP from 39+/-6 to 128+/-9 mm Hg (P<0.001), primarily because of peripheral vasoconstriction. In 3 dogs subjected to similar prolonged hemorrhagic shock, angiotensin II 180 ng. kg(-1). min(-1) had only a marginal effect on MAP (45+/-12 to 49+/-15 mm Hg). Plasma vasopressin was markedly elevated during acute hemorrhage but fell from 319+/-66 to 29+/-9 pg/mL before administration of vasopressin (P<0.01). CONCLUSIONS: Vasopressin is a uniquely effective pressor in the irreversible phase of hemorrhagic shock unresponsive to volume replacement and catecholamine vasopressors. Vasopressin deficiency may contribute to the pathogenesis of this condition.

Purification and partial characterization of canine angiotensinogen.

A procedure is described to isolate angiotensinogen (renin substrate) from canine plasma. The isolation procedure resulted in an 800-fold purification with a rate of recovery of approximately 12%. The purified protein has a specific activity of 24 micrograms of angiotensin I/mg protein. The amino terminal amino acid sequence of canine angiotensinogen was found to be identical to that of the horse but to differ from that of human and rat angiotensinogens. Canine angiotensinogen was heterogeneous with respect to molecular weight and isoelectric point. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pure angiotensinogen revealed two closely spaced bands with apparent molecular weights of 58,000 and 56,000. Chromatofocusing showed four isoforms: Peaks of pure angiotensinogen eluted at pH levels of 4.32, 4.23, 4.15, and 4.04. Isoelectric focusing confirmed the presence of four isoforms. Thus, the purification procedure identified two molecular weight forms and four isoforms of canine angiotensinogen. Isolation of the four isoforms will allow their characterization and the study of their physiological significance.

Urinary catecholamine metabolites distinguish different types of sympathetic neuronal dysfunction in patients with orthostatic hypotension.

Urinary excretion rates of the major norepinephrine metabolites, 3-methoxy-4-hydroxymandelic acid, 3-methoxy-4-hydroxy-phenylglycol and normetanephrine, were determined in 12 normal subjects and 23 patients with neurogenic orthostatic hypotension due to either multiple system atrophy [Shy-Drager Dyndrome (MSA)] or idiopathic orthostatic hypotension (IOH). There were striking and parallel decreases in all catecholamine metabolites in IOH consistent with loss of peripheral sympathetic nerves. Patients with MSA excreted greater amounts of the deaminated metabolites than did the patients with IOH, but most excreted equally low amounts of normetanephrine. The disproportionate decrease in excretion of normetanephrine by patients with MSA is consistent with observations in experimental animals that O-methylation is the primary metabolic route for active released norepinephrine, whereas deamination is the predominant metabolic route for intraneuronal degradation of the catecholamine. The similar proportional decreases in all catecholamine metabolites in patients with IOH (who have no central nervous system deficit) indicates that brain norepinephrine is a source of only a small fraction of urinary norepinephrine metabolites, including 3-methoxy-4-hydroxy-phenylglycol.

Effect of lithium therapy on glomerular filtration rate.

Patients taking lithium had a slightly higher serum creatinine concentration than controls. Creatinine concentration was independent of lithium level or therapy length, suggesting that lithium decreases glomerular filtration but that this effect is small, noncumulative, and of marginal clinical significance.

Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system.

Using a cell-based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0-200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV-deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell-based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin.

Cloning, expression, and characterization of mouse tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

Active site-inactivated factors VIIa, Xa, and IXa inhibit individual steps in a cell-based model of tissue factor-initiated coagulation.

Factors VIIa, Xa, and IXa play different roles in the initiation of tissue factor-dependent coagulation. The consequences of competing with the different enzymes were investigated, thereby examining the effects of inhibiting the initiation process at different steps. Active site-inactivated factors VIIa, Xa, and IXa (FVIIai, FXai, and FIXai, respectively) were added to various cell-based assays mimicking the individual steps in tissue factor-initiated coagulation. In an assay involving tissue factor-expressing monocytes, coagulation proteins and unactivated platelets, FVIIai and FXai inhibited platelet activation and thrombin generation while FIXai only inhibited thrombin generation. FVIIai inhibited factor Xa generation and subsequent thrombin generation on monocytes, while FXai inhibited thrombin generation on the monocytes as well as on the activated platelets. FIXai had no effect on factor Xa or thrombin generation on the monocytes, but inhibited factor Xa and subsequent thrombin generation on the activated platelets. FVIIai had no effect on the reactions taking place on the activated platelets. The data confirm a model where tissue factor/factor VIIa mediates factor Xa generation and subsequent prothrombin activation on the tissue factor-bearing cells. Thrombin then activates platelets, which serve as the physiologically important surface for large-scale thrombin generation.