Comparison of the Fixation Strengths of Screws between the Traditional Trajectory and the Single and Double Endplate Penetrating Screw Trajectories Using Osteoporotic Vertebral Body Models Based on the Finite Element Method.

STUDY DESIGN: This is a finite element (FE) study. PURPOSE: To compare the fixation strength of traditional trajectory (TT) and single and double endplate penetrating screw trajectories (SEPST/DEPST) to the osteoporotic vertebral body model based on the FE method. OVERVIEW OF LITERATURE: SEPST/DEPST have been developed to enhance the fixation strength in patients with diffuse idiopathic hyperostosis (DISH). This technique was also applied to patients with osteoporosis. However, determining the superiority of SEPST/ DEPST is difficult because of the heterogeneous patient backgrounds. METHODS: Twenty vertebrae (T12 and L1) from 10 patients with osteoporosis (two males and eight females; mean age, 74.7 years) were obtained to create the 10 FE models. First, a single screw was placed with TT and SEPST/DEPST, and the fixation strength was compared by axial pullout strength (POS) and multidirectional loading tests. Second, two screws were placed on the bilateral pedicles with TT and SEPST/DEPST, and the fixation force of the vertebrae in the constructs in flexion, extension, lateral flexion, and axial rotation was examined. RESULTS: SEPST and DEPST had 140% and 171% higher POS values than TT, respectively, and the DEPST result was statistically significant (p =0.007). The multidirectional fixation strength was significantly higher in DEPST and SEPST than in TT in the cranial, caudal, and medial directions (p <0.05) but not in the lateral direction (p =0.05). The vertebral fracture strength at the lower instrumented vertebra of the DEPST tended to be higher than that of TT. The vertebral motion angles in SEPST and DEPST were significantly smaller in lateral bending (p =0.02) and tended to be smaller in flexion and extension than in TT (p =0.13). CONCLUSIONS: This study may provide useful information for spine surgeons in deciding whether to choose the SEPS or DEPS technique for augmenting fixation in osteoporotic vertebral fracture surgery.

Evaluation of intraoperative coronal alignment using a computer-assisted rod bending system (CARBS) without intraoperative radiation exposure in adult spinal deformity surgery: a technical note and preliminary results.

PURPOSE: Intraoperative radiographs and fluoroscopy are used in adult spinal deformity (ASD) surgery to prevent postoperative coronal malalignment but with limited accuracy. Therefore, we applied a computer-assisted rod bending system (CARBS: Bendini®) for an intraoperative coronal alignment evaluation. The purpose of this study is to introduce this novel technique and validate its accuracy. METHODS: Fifteen ASD patients were included in the study. The heads of the bilateral S1 pedicle screws (S1), the S1 spinous process, and the bilateral greater trochanter (GT) and the C7 spinous process were recorded with CARBS for an intraoperative coronal alignment evaluation. The lines which connect the bilateral S1 and GT were used as references. The C7-center sacral vertical line (C7-CSVL) on the CARBS monitor was checked, and the C7-CSVL from the intraoperative CARBS recording and postoperative standing whole spine radiograph were compared. RESULTS: Intraoperative C7-CSVL with CARBS was 35.1 ± 31.6 mm when the S1 pedicle screws were used as the reference line and was 16.6 ± 17.8 mm when the GTs were used. Postoperative C7-CSVL by radiograph was 15.1 ± 16.5 mm. In addition, the intraoperative C7-CSVL with CARBS and the postoperative C7-CSVL showed a strong positive correlation in both GT (R = 0.86, p < 0.01) and in S1(R = 0.79, p < 0.01), with a better correlation found in GT than in S1. CONCLUSION: Intraoperative C7-CSVL with CARBS was found to be highly accurate in ASD surgery. Our results suggest that this novel technique can be useful as an alternative to intraoperative radiography and fluoroscopy and may reduce radiation exposure.

Visualization of Domain- and Concentration-Dependent Impact of Thrombomodulin on Differential Regulation of Coagulation and Fibrinolysis.

BACKGROUND: Thrombomodulin (TM) functions as a dual modulator-anticoagulant and antifibrinolytic potential-by the thrombin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated TAFI cleaves the C-terminal lysine of partially degraded fibrin and inhibits both plasminogen binding and its activation on the fibrin surface. We have reported previously that activated platelets initiate fibrin network formation and trigger fibrinolysis after the accumulation of tissue-type plasminogen activator and plasminogen. OBJECTIVE: To analyze the effects of domain-deletion variants of TM on coagulation and fibrinolysis at different concentrations. METHODS: Domain-deletion variants of TM, such as D123 (all extracellular regions), E3456 (minimum domains for thrombin-dependent activation of protein C and TAFI), and E456 (minimum domains for that of protein C but not TAFI), were used at 0.25 to 125 nM for turbidimetric assay to determine the clotting time and clot lysis time and to visualize fibrin network formation and lysis in platelet-containing plasma. RESULTS AND CONCLUSIONS: A low concentration of either D123 or E3456, but not of E456, prolonged clot lysis time, and delayed the accumulation of fluorescence-labeled plasminogen at the activated platelets/dense fibrin area due to effective TAFI activation. Conversely, only the highest concentrations of all three TM variants delayed the clotting time, though fibrin network formation in the vicinity of activated platelets was almost intact. TAFI activation might be affected by attenuation in thrombin activity after the clot formation phase. These findings suggest that the spatiotemporal balance between the anticoagulant and antifibrinolytic potential of TM is controlled in domain- and concentration-dependent manners.

Radiological Evaluation of Combined Anteroposterior Fusion with Vertebral Body Replacement Using a Minimally Invasive Lateral Approach for Osteoporotic Vertebral Fractures: Verification of Optimal Surgical Procedure.

The combined anteroposterior fusion with vertebral body replacement (VBR) using a wide footplate expandable cage with a minimally invasive lateral approach has been widely used for pseudoarthrosis after osteoporotic vertebral fractures. The purpose of this study is to evaluate the radiological results of combined anteroposterior surgery using VBR and to recommend the optimal procedure. Thirty-eight elderly patients were included in this study. The mean preoperative local kyphosis angle was 29.3°, and the mean correction loss angle was 6.3°. Cage subsidence was observed in ten patients (26.3%), and UIV or LIV fracture in twelve patients (31.6%). Patients with cage subsidence were compared to those without cage subsidence to determine the causal factors. The mean number of fixed vertebrae was 5.4 vertebrae with cage subsidence and 7.4 vertebrae without cage subsidence. In addition, to precisely clarify the optimal number of fixed vertebrae, those patients with two above-two below fixation were compared to those with less than two above-two below fixation, which revealed that the correction loss angle was significantly less in two above-two below fixation (p = 0.016). Based on these results, we recommend at least two above-two below fixation with VBR to minimize the correction loss angle and prevent cage subsidence.

Pre-administration of a carboxypeptidase inhibitor enhances tPA-induced thrombolysis in mouse microthrombi: Evidence from intravital imaging analysis.

INTRODUCTION: Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological treatment of choice in acute thrombotic events. However, a narrow therapeutic window and bleeding complications limit its use. We describe the role of carboxypeptidase inhibitor from potato tuber (PTCI), an inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), on Glu-plasminogen accumulation and microthrombus dynamics in vivo and demonstrate its influence on rt-PA-mediated thrombolysis. MATERIALS AND METHODS: In conjunction with real-time intravital two-photon excitation fluorescence microscopy, we produced and imaged laser-induced microthrombi in the mesenteric venules of Green Fluorescent Protein (GFP)-expressing mice. We examined microthrombus dynamics and thrombolysis patterns in vivo by measuring the changes in the fluorescence intensity of labeled Glu-plasminogen following administration of epsilon aminocaproic acid (EACA), PTCI, and rt-PA. RESULTS: PTCI enhanced Glu-plasminogen accumulation at the core of the thrombus by inhibiting TAFIa, while EACA inhibited this process. Exogenous rt-PA effectively triggered Glu-plasminogen activation within the thrombus and promoted thrombolysis. Administration of PTCI and rt-PA together showed no significant benefit on thrombolysis compared to rt-PA administration alone. However, early-phase systemic administration of PTCI before thrombolytic therapy by rt-PA expedited clot lysis as evidenced by significantly faster time to reach peak Glu-plasminogen fluorescence intensity and shorter time to achieve near-complete clot lysis (P = 0.014 and P = 0.003, respectively). CONCLUSIONS: PTCI potentiates rt-PA-mediated thrombolysis when administered early in acute thrombotic events. Further studies are warranted to explore the potential of TAFI inhibitors as adjunct agents in thrombolysis or thromboprophylaxis.

Metastatic intradural extramedullary spinal cord tumor from ovarian cancer: A case report with a literature review.

Context: Metastatic intradural extramedullary spinal cord tumors are extremely rare.Findings: A 76-year-old woman presented with intractable neck pain. Three years earlier, she had been treated for ovarian cancer with bilateral salpingo-oophorectomy. A year later, she underwent resection of a brain metastasis. Magnetic resonance imaging (MRI) showed an encapsulated intradural extramedullary mass at C4-C5. C4-C5 hemilaminectomy, tumor resection, and biopsy were performed. Histological examination of the resection revealed an adenocarcinoma. After surgery, her intolerable neck-shoulder pain was fully resolved, and she had no difficulties with daily living activities. However, two months later, she underwent gamma knife radiosurgery for the recurrent metastatic brain tumor, and four months later, she died from cachexia.Conclusion: Although cases of metastatic intradural extramedullary spinal tumors from ovarian cancer are extremely rare, their possibility should be considered in the differential diagnosis. A history of brain metastases and enhancement on T1-weighted MRI were helpful for making an accurate diagnosis.

Demonstration of Three Distinct High-Molecular-Weight Complexes between Plasminogen Activator Inhibitor Type 1 and Tissue-Type Plasminogen Activator.

BACKGROUND: Details of the molecular interaction between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) remain unknown. METHODS AND RESULTS: Three distinct forms of high-molecular-weight complexes are demonstrated. Two of the forms were detected by mass spectrometry. The high molecular mass detected by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was 107,029 Da, which corresponds to the sum of molecular masses of the intact tPA (65,320 Da) and the intact PAI-1 (42,416 Da). The lower molecular mass was 104,367 Da and is proposed to lack the C-terminal bait peptide of PAI-1 (calculated mass: 3,804 Da), which was detected as a 3,808 Da fragment. When the complex was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), only a single band was observed. However, after treatment by SDS and Triton X-100, two distinct forms of the complex with different mobilities were shown by SDS-PAGE. The higher molecular weight band demonstrated specific tPA activity on fibrin autography, whereas the lower molecular weight band did not. Peptide sequence analysis of these two bands, however, unexpectedly revealed the existence of the C-terminal cleavage peptide in both bands and its amount was less in the upper band. In the upper band, the sequences corresponding to the regions at the interface between two molecules in its Michaelis intermediate were diminished. Thus, these two bands corresponded to distinct nonacyl-enzyme complexes, wherein only the upper band liberated free tPA under the conditions employed. CONCLUSION: These data suggest that under physiological conditions a fraction of the tPA-PAI-1 population exists as nonacylated-enzyme inhibitor complex.

Fibrinolysis-resistant carbonylated fibrin detected in thrombi attached to the vascular wall of abdominal aortic aneurysms.

In this study, we investigated how carbonylation of fibrinogen by acrolein modified its indispensable function to enhance fibrinolysis after being converted to fibrin and contributed to generating a fibrinolysis-resistant fibrin clot. Acrolein-treated fibrinogen was subjected to tissue plasminogen activator-induced fibrinolysis assay and the effect of lysine residue carbonylation in fibrinogen on fibrinolysis was analyzed. The acrolein-treated fibrinogen-derived fibrin clot appeared more resistant to fibrinolysis and the N-acetyl 3-formyl-3,4-dehydropiperidino (FDP)-Lysine levels in the lysed solution were positively correlated with the duration of clot lysis. The lysine analog 6-amino hexanoic acid (6AHA), which mimics the C-terminal lysine of fibrin, was carbonylated and its enhancing effect on Glu1-plasminogen activation was evaluated. After incubation with acrolein, 6AHA was converted to N-acetyl FDP-6AHA, losing its ability to enhance Glu1-plasminogen activation. These results suggest that fibrinogen carbonylation by acrolein to generate N-acetyl FDP-Lysine resulted in the generation of fibrinolysis-resistant fibrin by attenuating the C-terminal lysine-dependent activation of the Glu1-plasminogen. In abdominal aortic aneurysms, fibrin(ogen) containing the acrolein adduct N-acetyl FDP-Lysine was detected in the vascular wall-attached thrombi. These results suggest that this mechanism is likely involved in the modification of fibrinolysis-resistant thrombi and to their persistence for a long period.

Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system.

Our previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their procoagulant functions, platelets enhanced profibrinolytic potential by augmenting the accumulation of tissue-type plasminogen activator (tPA) and plasminogen, in vivo in a murine microthrombus model, and in vitro in a platelet-containing plasma clot model. To clarify the role of thrombin-activatable fibrinolysis inhibitor (TAFI), which regulates coagulation-dependent anti-fibrinolytic potential, we analyzed tPA-induced clot lysis times in platelet-containing plasma. Platelets prolonged clot lysis times in a concentration-dependent manner, which were successfully abolished by a thrombomodulin-neutralizing antibody or an activated TAFI inhibitor (TAFIaI). The results obtained using TAFI- or factor XIII-deficient plasma suggested that TAFI in plasma, but not in platelets, was essential for this prolongation, though its cross-linkage with fibrin was not necessary. Confocal laser scanning microscopy revealed that fluorescence-labeled plasminogen accumulated on activated platelet surfaces and propagated to the periphery, similar to the propagation of fibrinolysis. Plasminogen accumulation and propagation were both enhanced by TAFIaI, but only accumulation was enhanced by thrombomodulin-neutralizing antibody. Labeled TAFI also accumulated on both fibrin fibers and activated platelet surfaces, which were Lys-binding-site-dependent and Lys-binding-site-independent, respectively. Finally, TAFIaI significantly prolonged the occlusion times of tPA-containing whole blood in a microchip-based flow chamber system, suggesting that TAFI attenuated the tPA-dependent prolongation of clot formation under flow. Thus, activated platelet surfaces are targeted by plasma TAFI, to attenuate plasminogen accumulation and fibrinolysis, which may contribute to thrombogenicity under flow.

The Role of Animal Models in Elucidating the Etiology and Pathology of Abdominal Aortic Aneurysms: Development of a Novel Rupture Mechanism Model.

Existing animal models do not replicate all aspects of abdominal aortic aneurysms (AAAs), including the rupture mechanisms. From histopathological analyses conducted in humans, it has been found that the vasa vasorum of the AAA wall is the starting point of circulatory failure and that bulging and dilatation of the abdominal aorta occurs through inflammation and tissue degeneration. We created a new animal model (the hypoperfusion-induced model) of AAAs. In this study, we describe the current animal models of AAAs and present the utility of our new model of AAAs.

Recognition of Plasminogen Activator Inhibitor Type 1 as the Primary Regulator of Fibrinolysis.

The fibrinolytic system consists of a balance between rates of plasminogen activation and fibrin degradation, both of which are finely regulated by spatio-temporal mechanisms. Three distinct inhibitors of the fibrinolytic system that differently regulate these two steps are plasminogen activator inhibitor type-1 (PAI-1), α2-antiplasmin, and thrombin activatable fibrinolysis inhibitor (TAFI). In this review, we focus on the mechanisms by which PAI-1 governs total fibrinolytic activity to provide its essential role in many hemostatic disorders, including fibrinolytic shutdown after trauma. PAI-1 is a member of the serine protease inhibitor (SERPIN) superfamily and inhibits the protease activities of plasminogen activators (PAs) by forming complexes with PAs, thereby regulating fibrinolysis. The major PA in the vasculature is tissue-type PA (tPA) which is secreted from vascular endothelial cells (VECs) as an active enzyme and is retained on the surface of VECs. PAI-1, existing in molar excess to tPA in plasma, regulates the amount of free active tPA in plasma and on the surface of VECs by forming a tPA-PAI-1 complex. Thus, high plasma levels of PAI-1 are directly related to attenuated fibrinolysis and increased risk for thrombosis. Since plasma PAI-1 levels are highly elevated under a variety of pathological conditions, including infection and inflammation, the fibrinolytic potential in plasma and on VECs is readily suppressed to induce fibrinolytic shutdown. A congenital deficiency of PAI-1 in humans, in turn, leads to life-threatening bleeding. These considerations support the contention that PAI-1 is the primary regulator of the initial step of fibrinolysis and governs total fibrinolytic activity.

Hand Dexterity Impairment in Patients with Cervical Myelopathy: A New Quantitative Assessment Using a Natural Prehension Movement.

Cervical myelopathy (CM) caused by spinal cord compression can lead to reduced hand dexterity. However, except for the 10 sec grip-and-release test, there is no objective assessment system for hand dexterity in patients with CM. Therefore, we evaluated the hand dexterity impairment of patients with CM objectively by asking them to perform a natural prehension movement. Twenty-three patients with CM and 30 age-matched controls were asked to reach for and grasp a small object with their right thumb and index finger and to subsequently lift and hold it. To examine the effects of tactile afferents from the fingers, objects with surface materials of differing textures (silk, suede, and sandpaper) were used. All patients also underwent the Japanese Orthopedic Association (JOA) test. Preoperative patients showed significantly greater grip aperture during reach-to-grasp movements and weaker grip force than controls only while attempting to lift the most slippery object (silk). Patients, immediately after surgery, (n = 15) tended to show improvements in the JOA score and in reaction time and movement time with respect to reaching movements. Multiple regression analysis demonstrated that some parameters of the prehension task could successfully predict subjective evaluations of dexterous hand movements based on JOA scores. These results suggest that quantitative assessments using prehension movements could be useful to objectively evaluate hand dexterity impairment in patients with CM.

Hypoperfusion of the Aortic Wall Secondary to Degeneration of Adventitial Vasa Vasorum Causes Abdominal Aortic Aneurysms.

BACKGROUND: An abdominal aortic aneurysm (AAA), which affects approximately 10% of Japanese men aged ≥ 65 years, is frequently associated with hypertension, dyslipidemia, and other lifestyle- related diseases. The development of an AAA is attributed to chronic inflammation concomitant with arteriosclerotic changes. However, an accurate pathomechanism associated with AAA remains uncertain, and questions such as why only a particular group/percentage of patients with arteriosclerosis develop aneurysms and how diabetes suppresses aneurysm development remain unanswered. OBJECTIVE: We examined a novel mechanism to develop AAA based on histopathological findings following analysis of the human AAA tissues. Additionally, based on these findings, we developed a new animal model of AAA, in which the histopathological characteristics are similar to human AAA tissue. RESULTS: Recently, we identified stenosis of the vasa vasorum (VV) in aortic segments showing dilatation. The aorta is the largest artery in our circulatory system. Under physiological conditions, the inner layer of the aorta is nourished via direct diffusion of nutrients from the luminal blood flow, whereas the outer adventitia is primarily perfused by the VV. Therefore, hypoperfusion of the VV induces hypoxia and subsequent inflammation and tissue degeneration of the aortic wall, resulting in aneurysm formation. Based on these findings, we established an AAA animal model by reducing the blood flow through the VV to the aortic wall. AAA was successfully reproduced in our animal model. Histopathological findings in this model were indistinguishable from those observed in humans, and pronounced abnormality in lipid composition in blood vessel adventitia was also observed. CONCLUSION: Thus, hypoperfusion of the aortic wall appeared to be sufficient to cause inflammationinduced AAA. These findings may provide potential targets for novel therapeutics for the management of an AAA.

Ectopic Expression of PCSK9 by Smooth Muscle Cells Contributes to Aortic Dissection.

BACKGROUND: Acute aortic dissection (AAD) is a common disease among the elderly. Although several risk factors of AAD have been reported, the molecular mechanism underlying AAD development remains to be elucidated. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases low-density lipoprotein cholesterol levels in blood by preventing its clearance. Therefore, PCSK9 inhibition is a promising therapeutic approach to treat cardiovascular diseases (CVDs). The objective of this study was to elucidate the role of PCSK9 in the pathogenesis of AAD. METHODS: We used fluorescence immunohistochemistry to assess PCSK9 expression in aortic tissues resected from 10 AAD patients and in the normal aorta from 5 autopsy samples as well as in spontaneously hyperlipidemic apolipoprotein E-deficient mice used as an experimental AD model. RESULTS: We revealed a characteristic distribution pattern of PCSK9 in atherosclerotic plaques and the degenerated tunica media in AAD tissues, which was rarely observed in normal aortic tissues. Furthermore, PCSK9 was notably expressed around calcification areas formed by vascular smooth muscle cells, especially those of the synthetic phenotype. The results obtained in the animal model were consistent with PCSK9 expression in AAD tissues. CONCLUSIONS: Our findings suggest that PCSK9 overexpression in the aorta may promote AAD. This study adds to the growing body of evidence supporting the use of PCSK9 inhibitors for the management of CVDs.

Elevated Plasma Levels of LDL Cholesterol Promote Dissecting Thoracic Aortic Aneurysms in Angiotensin II-Induced Mice.

BACKGROUND: Plasma low-density lipoprotein (LDL) cholesterol is implicated in abdominal aorta (AA) and aortic dissection (AD); however, its role in the pathogenesis of AA and AD, a disease with a high mortality rate, is unknown. The existing animal models such as apolipoprotein E-deficient (Apoe-/-) mice cannot reproduce all the conditions of AA/AD, including elevated LDL-cholesterol levels and spontaneous atheroma formation; therefore, a more reliable in vivo model is required. Here, we analyzed angiotensin II (Ang II)-induced mice with combined deficiency of the LDL receptor and the catalytic component of the apolipoprotein B-edisome complex (Ldlr-/-/Apobec1-/- [WKO]) to understand AA formation and AD occurrence in relation to plasma lipid composition. METHODS: AAs and ADs were created in 18- to 22- week-old male Apoe-/- and Ldlr-/-/Apobec1-/- mice by Ang II infusion. Immunostaining allowed assessment of smooth muscle cells and mural monocytes/macrophages. RESULTS: Ldlr-/-/Apobec1-/- mice had elevated LDL-cholesterol levels characteristic for human type IIa hyperlipidemia, resulting in atherogenesis, which promoted mortality, AA formation, and AD development. Interestingly, variations in the distribution of atheromas and inflammatory sites between Apoe-/- and Ldlr-/-/Apobec1-/- mice depending on lipid profiles resulted in differences in AA formation and AD occurrence in the thoracic aorta. CONCLUSIONS: Our results indicate the presence of a pathogenic pathway involving serum lipid composition that plays a key role in AA formation and AD occurrence in Ang II-induced mice.

Lysophosphatidylcholine Acyltransferase-3 Expression Is Associated with Atherosclerosis Progression.

Free arachidonic acid (AA) is an important precursor of lipid mediators such as leukotrienes and prostaglandins that induces inflammation and is associated with atherosclerosis progression. Recent studies have shown that lysophosphatidylcholine acyltransferase-3 (LPCAT3) converts lysophosphatidylcholine (LPC) and free AA into phosphatidylcholine (PC)-containing AA (arachidonyl-PC) and thereby can regulate intracellular free-AA levels. However, the association between LPCAT3 and atherosclerosis remains to be established. In this study, we analyzed human and mouse atherosclerotic tissues to gain insight into the arachidonyl-PC metabolism involving LPCAT3 using imaging mass spectrometry. The data revealed a complementary distribution of arachidonyl-PC and LPC in human atherosclerotic tissues with arachidonyl-PC decreasing and LPC increasing as atherosclerosis progressed. Furthermore, we found a homologous distribution of LPCAT3 expression and arachidonyl-PC based on atherosclerotic progression. In contrast, in ApoE-deficient mice, atherosclerosis increased both arachidonyl-PC accumulation and LPCAT3 expression. Taken together, these findings suggest that the regulation of LPCAT3 expression might be associated with atherosclerotic progression in humans.

Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

Activities of wild-type and variant tissue-type plasminogen activators retained on vascular endothelial cells.

We reported that tissue-type plasminogen activator (tPA) secreted from vascular endothelial cells (VECs) is retained on the cell surface and effectively evokes both plasminogen activation and fibrin clot dissolution (fibrinolysis) on VECs. Here, to evaluate possibly different behaviors of variants of tPA, we quantitatively assessed these two events separately using green fluorescent protein (GFP)-conjugated tPA in cultured human VECs. The amount of secreted wild-type tPA-GFP correlated well with both the activities of plasminogen activation (r = 0.66) and fibrinolysis (r = -0.93). A variant of tPA-GFP, with a lower affinity to the surface of VECs but higher affinity to fibrin, showed higher fibrinolysis and lower plasminogen activation activity compared to the wild-type.

Bidirectional functions of thrombin on fibrinolysis: Evidence of thrombin-dependent enhancement of fibrinolysis provided by spontaneous plasma clot lysis.

Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We demonstrated that the euglobulin clot lysis time (ECLT) was shortened by endogenously generated thrombin as a result of the inactivation of plasminogen activator inhibitor type 1 (PAI-1). In contrast, thrombin suppressed fibrinolytic activity through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Here, using three different clot lysis assays of the ECLT, the tissue plasminogen activator supplemented plasma clot lysis time (tPA-PCLT) and the spontaneous plasma clot lysis time (s-PCLT), we analyzed how the coagulation process modifies fibrinolysis. The ECLT was shortened by exogenously supplemented thrombin in a dose-dependent manner in the absence of calcium ion (Ca(++)), whereas this shortening was not observed in the presence of Ca(++) where endogenous prothrombin was effectively activated to thrombin. This shortening was also not observed for the tPA-PCLT, in which tPA is supplemented in excess and PAI-1 activity is mostly lost. On the contrary, thrombin dose-dependently prolonged the tPA-PCLT, which was mostly abolished by inhibitors of carboxypeptidase and activated FXIII, suggesting that the prolongation is TAFI- and Factor XIII-dependent. The s-PCLT was shortened when thrombin generation was boosted by supplementing tissue factor and phosphatidylserine together with Ca(++), which was more apparent in the presence of inhibitors of activated FXIII and activated TAFI. Thus, thrombin appeared to express its enhancing effect on fibrinolysis even in plasma, in addition to its inhibiting effect. These bidirectional functions of thrombin on fibrinolysis seem to take place on demand under different environments to maintain adequate vascular blood flow.

Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.